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The present invention provides application of the Artesunate as immunologic adjuvant in preparation rabies vacciness, and present invention firstly provides artemisinin derivative Artesunate (ART) is used as rabies vacciness adjuvant.The present invention passes through experimental studies have found that artemisinin derivative Artesunate (ART) is as rabies vacciness adjuvant can significantly increase its immunogenicity, can be used as new semple type rabies vaccine candidate adjuvant.The Artesunate (ART) of vaccine effect studies have shown that relatively low-dose of the invention can promote the immune effect of rabies vacciness, can reduce the cost of production vaccine.Vaccine effect studies have shown that artemisinin derivative Artesunate (ART) of the invention can generate better protection as rabies vacciness adjuvant. 本发明提供青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用，本发明首次提出将青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂。本发明通过实验研究发现青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂能显著增强其免疫原性，可以作为新型狂犬病疫苗候选佐剂。本发明的疫苗效果研究显示，较低剂量的青蒿琥酯(ART)能促进狂犬病疫苗的免疫效果，可以降低制作疫苗的成本。本发明的疫苗效果研究显示，青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂能产生更好的保护力。 Application of the Artesunate as immunologic adjuvant in preparation rabies vacciness Technical field The invention belongs to molecular biosciences field of immunology.It is being prepared more particularly, to Artesunate as immunologic adjuvant Application in rabies vacciness. Background technique Hydrophobin (Rabies virus, RABV) is the strong people beast caused with the characteristics of infecting central nervous system Suffer from the rabic cause of disease of infectious disease altogether, once morbidity, lethality are up to 100%.China is rabic district occurred frequently, number of the infected It is only second to India, is in second place of the world.With the increase of domestic pets, the rabic control in China human world and prevention and treatment are still appointed Weight road is remote.However rabies pathogenesis is also imperfectly understood so far, also without treating rabic specific drug, so prevention is mad The most effective measure of dog disease is timely vaccine inoculation. Currently, be both at home and abroad the protein formulation purified for the rabies vacciness of the mankind, due to a lack of corresponding adjuvant, first It is secondary it is immune need to inject 3-5 needle, take one month.As for animal rabies vaccine, import inactivated vaccine have using it is safe, be easy to The advantages such as preservation, pollution-free danger.Although China's approved several moneys inactivated vaccines with independent intellectual property rights at present, because The factors such as higher of production-scale limitation and production cost, these inactivated vaccines hair more backward for Asia and African economy Price is too high for area in exhibition, limits them in the use of developing country, and most of economically developed city is still Preferred import inactivated vaccine.Domestic inactivated vaccine needs for animals further reduce the cost. Summary of the invention The defects of higher the technical problem to be solved by the present invention is to overcome existing vaccine cost and deficiency, provide Artesunate As application of the immunologic adjuvant in preparation rabies vacciness. The object of the present invention is to provide Artesunate (referred to as (ART)) as active constituent in preparation rabies vacciness adjuvant In application. By ART and hydrophobia strain mixed immunity mouse, immune in mouse of enhancing inactivation hydrophobin is answered It answers.And the recruitment evaluation of vaccine after identifying and mixing with vaccine, experimental result hair are further carried out as vaccine adjuvant to ART Existing, Artesunate can significantly increase the immunogenicity of rabies vacciness, can be used as new semple type rabies vaccine candidate adjuvant. In one embodiment of this invention, the Artesunate is as sole active agent. The present invention also provides a kind of rabies vacciness adjuvant, the rabies vacciness adjuvant includes Artesunate. In one embodiment of this invention, the Artesunate is as sole active agent. The present invention also provides application of the Artesunate in preparation rabies vacciness, and the Artesunate is as adjuvant. The present invention also provides a kind of vaccine compositions, and it includes the hydrophobin strains and Artesunate of inactivation. In one embodiment of this invention, the hydrophobin strain of the inactivation is selected from CVS-11 strain, rHEP-dG poison Strain, SAD plants, CTN-1 plants, PV plants, at least one of FluryLEP plants.Certainly, above-mentioned strain, all rabies diseases are not limited to Poison strain is suitable for the present invention. In one embodiment of this invention, in the vaccine composition hydrophobin strain and Artesunate amount ratio It is 0.9 × 105~1.1 × 105The μ of FFU:120~130 g. In one embodiment of this invention, the content of hydrophobin strain is 1.0 × 10 in the vaccine composition5~ 1.1×105The μ of FFU:120~130 g. In one embodiment of this invention, the dosage of Artesunate is 5 μ g/g the weight of animals in the vaccine composition.It is green The dosage of artemisic succinate is determined according to the weight of animals such as mouse, and by taking mouse as an example, actual use concentration is 5 μ g/g mouse weights, If every mouse weight is about 25g, the dosage of Artesunate is about 125 μ g. In one embodiment of this invention, further include dimethyl sulfoxide (DMSO), other solvents can also be used, DMSO exists When dissolving the reagent of some good water solubilities, solute effect is more preferable. In one embodiment of this invention, Artesunate (g) in the vaccine composition: dimethyl sulfoxide (mL)=1:(8 ~12), preferably 1:10. It in one embodiment of this invention, further include PBS buffer solution.PBS buffer solution has effects that physiological saline, in stabilization More advantageous, pH 7.20-7.40 in terms of albumen. In one embodiment of this invention, by volume, Artesunate in the vaccine composition: PBS buffer solution=1: (90~110), preferably 1:100. The present invention also provides application of the Artesunate in preparation rabies vacciness.The rabies vacciness is for preventing or controlling Treat people or the rabies of other animals. Optionally, the Artesunate is as adjuvant. Term " adjuvant " is the pharmacy or immunological reagent or composition for changing other agent efficacies, refer in a broad sense from Body does not provide wide spectrum substance that is immune, but can increasing the immunogenicity of the antigen of co-administration.Adjuvant can be added to vaccine In, increase the quantity and lasting protection of antibody by increasing immune response, to reduce the external of injection to the maximum extent Substance.Adjuvant can also be used for improving the effect of vaccine, be changed by helping to the immune anti-of certain types of immune system cell It answers, for example, according to the purpose of vaccine, by activating T cell rather than the B cell of secretory antibody.Therefore, adjuvant can be advantageously Adjust cytokine-expressing/secretion, antigen presentation, type of immune response etc..In the present invention, the situation of the term refers to work For the compound or composition of the carrier or auxiliary substance of immunogene and/or other drugs reactive compound. Wherein above-mentioned " antigen ", which is typically meant that, can be identified by immune system and can for example be adapted to by being formed to be used as Property immune response a part antibody or antigen specific T-cell come trigger antigen specific immune reaction substance.Antigen It is divided into two classes: comlete antigen and incomplete antigen according to property.Comlete antigen (complete antigen) abbreviation antigen, is one The existing immunogenicity of class, and have immunoreactive substance, such as most protein, bacterium, virus, bacterial exotoxin and intestines poison Element etc..Incomplete antigen, that is, haptens (hapten) is that only have immunoreactivity, and the substance of non-immunogenicity, such as absolutely mostly The hydrolysate and all lipoids of number polysaccharide (capsular polysaccharide of such as pneumococcus), capsular polysaccharide. The invention has the following advantages: Present invention firstly provides artemisinin derivative Artesunate (ART) is used as rabies vacciness adjuvant. The present invention passes through experimental studies have found that artemisinin derivative Artesunate (ART) can be shown as rabies vacciness adjuvant The immunogenicity for writing enhancing rabies vacciness, can be used as new semple type rabies vaccine candidate adjuvant. The Artesunate (ART) of vaccine effect studies have shown that relatively low-dose of the invention can promote exempting from for rabies vacciness Epidemic disease effect can reduce the cost of production vaccine. Vaccine effect studies have shown that artemisinin derivative Artesunate (ART) of the invention is used as rabies vacciness adjuvant Better protection can be generated. Detailed description of the invention Fig. 1 is shown as a kind of chemical formula of artemisinin derivative Artesunate (ART). Fig. 2, which is shown as ART, influences statistical chart to KM mouse weight, while setting up PBS control group. Peripheral blood anti-rabies virus neutralizing antibody water after mouse is immunized in the CVS-11 that Fig. 3 is shown as combining ART inactivation Flat statistical chart, while setting up PBS control group (P < 0.05 *). Peripheral blood anti-rabies virus neutralizing antibody water after mouse is immunized in the rHEP-dG that Fig. 4 is shown as combining ART inactivation Flat statistical chart, while setting up PBS control group (P < 0.05 *). Fig. 5 is shown as the survival rate statistical chart of CVS-11 attack mouse after ART is immunized mouse 14 days as vaccine adjuvant. Specific embodiment The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art Agent, method and apparatus. Unless stated otherwise, following embodiment agents useful for same and material are commercially available. Currently, the World Health Organization is recommended to use inactivation rabies vacciness according to its safety, in order to develop it is more effective and Cheaper inactivated vaccine, we, which can take, improves the means such as virus titer or the better adjuvant of searching. Fig. 1 is shown as the chemical formula of artemisinin derivative Artesunate (ART) used by following embodiment, Artesunate (ART) No. CAS is 88495-63-0, molecular formula: C19H28O8, molecular weight: 384.42, No. EINECS: 618-170-5. Artesunate used in following embodiment is purchased from TCI AmericaPortland company (Tokyo chemical conversion industry Co., Ltd.). Artemisinin derivative Artesunate (ART) adjuvant in following embodiment, is the CVS-11 or rHEP-dG with inactivation Strain is mixed with ART, forms rabies virus vaccine. Specifically: ART is initially dissolved in DMSO, and the CVS-11 in PBS with inactivation is dissolved in when then using in vivo Or rHEP-dG hydrophobin mixing.According to Artesunate (g): DMSO (mL)=1:10 ratio, the i.e. Artesunate of 1g are molten In 10mL DMSO, the Artesunate being then dissolved in DMSO is redissolved in PBS buffer solution solution, Artesunate and PBS The volume ratio of buffer is 1:100, and PBS buffer solution is phosphate buffer, pH 7.2-7.4. Use the ART as adjuvant, rabies vacciness induction can be promoted to generate in higher anti-rabies virus and anti- Body, and better protecting effect is generated, reduce the cost of Canine vaccine. Specifically, in the immunologic adjuvant, Artesunate is the dosage and 1.0 × 10 by 5 μ g/g mouse weights5FFU inactivation CVS-11 or rHEP-dG mixing, by right leg gastrocnemius intramuscular injection be immunized mouse after collect peripheral blood, utilize neutralize experiment Study anti-rabies virus neutralize antibody titers. In addition, artemisinin derivative Artesunate (ART) also exists in the application as vaccine adjuvant or in terms of preparing vaccine Within protection scope of the present invention. Artemisinin derivative Artesunate (ART) is initially dissolved in DMSO, is dissolved in PBS when then using in vivo In, it is used after being mixed with CVS-11 the or rHEP-dG hydrophobin of inactivation.The immunologic adjuvant Artesunate is by 5 μ g/g Dosage is mixed with the CVS-11 of inactivation or rHEP-dG, collects serum after mouse is immunized by right leg gastrocnemius intramuscular injection, is used Neutralize experiment detection anti-rabies virus neutralize antibody titers.The present invention is also to ART side effect, ART Adjuvanted vaccines immunogenicity It is studied with the biological characteristics such as malicious protection are attacked, to assess the safety and effect of its as vaccine adjuvant.The results show that ART will not generate significant side effect as new vaccine adjuvant in Mice Body, and ART can significantly increase vaccine as adjuvant Immunogenicity promotes vaccine-induced body to generate the anti-rabies virus neutralizing antibody with Vaccine effectiveness, can preferably protect Protect the attack that body resists lethal rabies virus. In terms of specific research method, the present invention utilizes immunology the relevant technologies, has studied in Mice Body ART as novel The recruitment evaluation of vaccine adjuvant. Embodiment 1 The research that novel rabies virus vaccine adjuvant ART influences mouse weight SPF grade Kunming (KM) mouse of 3-4 week old is divided into 2 groups, i.e. ART group and PBS control group, every group comprising 6 small Mouse.ART is injected with the dosage of 5 μ g/g by the right leg gastrocnemius muscle of mouse, and control group injects equivalent PBS.It is every after administration The weight of its detection mouse, monitors 15 days altogether.By the weight before respectively injecting on the weight ratio of all mouse, weight ratio is studied Example situation of change.As shown in Figure 2, the results showed that, ART not will lead to treatment mouse weight and mitigate, this is similar to PBS treatment.More Importantly, there is not abnormal symptom in treatment mouse.Therefore, the ART treatment that dosage is 5 μ g/g is safe to mouse. Embodiment 2 Artesunate (ART) in the intracorporal immunogenicity of KM mouse and attacks malicious Protective strategy to rabies vacciness 1, experimental method CVS-11 and rHEP-dG are inactivated with UV respectively.Then ART is mixed with the CVS-11 of inactivation or rHEP-dG, PBS is used as vehicle control.The SPF grade kunming mouse of 6~7 week old is divided into 4 groups, i.e., ART+CVS-11 group, PBS+CVS-11 group, ART+rHEP-dG group and PBS+rHEP-dG control group, every group includes 6 mouse.The right leg gastrocnemius injection of every mouse of experimental group 1.0×105The hydrophobin of FFU inactivation and the mixture of ART, the volume of mixture are 200 μ L, and ART's contains in mixture Measuring is 5 μ g ART/g mouse weights, Artesunate (g): dimethyl sulfoxide (mL)=1:10, by volume, Artesunate: PBS Buffer=1:100, control group intramuscular injection penetrate 1.0 × 105The hydrophobin of FFU inactivation and PBS mixture.7th after immune It and the 14th day acquisition peripheral blood, and by fluorescence antibody virus neutralization (FAVN) testing inspection serum anti-rabies virus And antibody level, rabies standard serum are purchased from the World Health Organization (WHO), detection method is in strict accordance with world animal health Organize the standard practice instructions of (World Organisation for Animal Health, OIE). 2, experimental result As shown in Figure 3, the results showed that, the hydrophobin that mouse generates is immunized with the CVS-11 joint ART of inactivation and neutralizes Antibody level is significantly higher than PBS control group.As shown in figure 4, the mad dog that mouse generates is immunized with the rHEP-dG joint ART of inactivation Sick virucidin's level is significantly higher than PBS control group. Fig. 3 and Fig. 4's the result shows that, when ART is applied to rabies vacciness as adjuvant, it is not limited to a certain specific Inactivation hydrophobin strain, but can be adapted for the hydrophobin strain of various inactivations. 3, further, in order to verify after mouse obtains high level immunity whether there is protection to strong virus attack, In CVS-11 strain living is passed through intramuscular injection infecting mouse by 14d after immune, while being arranged without immune rabies vacciness Control group, the morbidity and death condition of continuous 3 weeks observation mouse, records survival rate daily.As shown in Figure 5, the results showed that, it uses ART as adjuvant mouse than there is higher survival rate with the mouse of PBS control. The above results show that artemisinin derivative Artesunate (ART) enhances inactivation rabies epidemic disease when being used as adjuvant The immune response of seedling. The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention. Claims (9) Hide Dependent 1. application of the Artesunate as active constituent in preparation rabies vacciness adjuvant. 2. rabies vacciness adjuvant according to claim 1, which is characterized in that the Artesunate as sole active at Point. 3. a kind of rabies vacciness adjuvant, which is characterized in that the rabies vacciness adjuvant includes Artesunate. 4. rabies vacciness adjuvant according to claim 3, which is characterized in that Artesunate is as sole active agent. 5. application of the Artesunate in preparation rabies vacciness, which is characterized in that the Artesunate is as adjuvant. 6. a kind of vaccine composition, which is characterized in that it includes the hydrophobin strains and Artesunate of inactivation. 7. vaccine composition according to claim 6, which is characterized in that the hydrophobin strain of the inactivation is selected from At least one of CVS-11 strain, rHEP-dG strain. 8. vaccine composition according to claim 6, which is characterized in that hydrophobin strain in the vaccine composition Amount ratio with Artesunate is 0.9 × 105~1.1 × 105The μ of FFU:120~130 g. 9. vaccine composition according to claim 8, which is characterized in that hydrophobin strain in the vaccine composition Amount ratio with Artesunate is 1.0 × 105~1.1 × 105The μ of FFU:120~130 g. 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Dhungyel et al.2014Footrot vaccines and vaccination Marcinkiewicz et al.2018Eliminating factor H-binding activity of Borrelia burgdorferi CspZ combined with virus-like particle conjugation enhances its efficacy as a Lyme disease vaccine CN107073097A2017-08-18Double adjuvant vaccine compositions, preparation and use CN107296955B2021-11-23Immunogenic bordetella bronchiseptica compositions Relyveld et al.1983[3] Preparation of vaccines by the action of glutaraldehyde on toxins, bacteria, viruses, allergens, and cells KR0162074B11998-12-01Solutions containing antigen and zinc hydroxide or iron hydroxide as an adjuvant and processes for preparing such solutions KR19980025002A1998-07-06Improved Inactive Vaccine TWI648063B2019-01-21Composition and method for immunization against C. difficile CN110507819A2019-11-29Application of the Artesunate as immunologic adjuvant in preparation rabies vacciness CN100572532C2009-12-23A kind of duck plague vaccine and special strains thereof Grubhofer1995An adjuvant formulation based on N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine with dimethyldioctadecylammonium chloride and zinc-L-proline complex as synergists Sampath et al.2005An immunogenicity study of a newly introduced purified Vero cell rabies vaccine (Abhayrab) manufactured in India Hosty et al.1959Human antirabies gamma globulin Panse et al.1964Passive immunity in experimental cholera CN110179975B2021-01-08Vegetable oil vaccine adjuvant and preparation method and application thereof CN101717779B2012-02-01Fusion protein suitable for anthrax antitoxin and vaccines WO2016036503A12016-03-10Antigen compositions and methods of use for treating extraintestinal pathogenic e. coli infections DK2376113T32014-12-08PREPARATIONS AND DOSAGE REGIMES WHICH INCLUDES A CLOSTRIDIUM VACCINE AND levamisole US6905712B22005-06-14Vaccine adjuvants comprising ginseng plant extract and added aluminum salt Priority And Related Applications Priority Applications (1) ApplicationPriority dateFiling dateTitle CN201910745298.1A2019-08-132019-08-13Application of artesunate as immunologic adjuvant in preparation of rabies vaccine Applications Claiming Priority (1) ApplicationFiling dateTitle CN201910745298.1A2019-08-13Application of artesunate as immunologic adjuvant in preparation of rabies vaccine https://patents.google.com/patent/CN110507819A/en 青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用 技术领域 本发明属于分子生物免疫学领域。更具体地，涉及青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用。 背景技术 狂犬病病毒(Rabies virus，RABV)是引起以侵染中枢神经系统为特点的烈性人兽共患传染病狂犬病的病原，一旦发病，致死率高达100％。我国是狂犬病的高发区，发病人数仅次于印度，高居世界第二位。随着家养宠物的增加，我国人间狂犬病的控制和防治依然任重道远。然而狂犬病致病机理至今还不完全清楚，也没有治疗狂犬病的特效药，所以预防狂犬病最有效的措施是及时接种疫苗。 目前，国内外用于人类的狂犬病疫苗为纯化的蛋白制剂，因缺乏相应的佐剂，第一次免疫需注射3-5针，需时一个月。至于动物狂犬病疫苗，进口灭活疫苗具有使用安全、易于保存、无污染危险等优势。我国目前虽然已批准了几款具有自主知识产权的灭活疫苗，但因生产规模的限制和生产成本的偏高等因素，这些灭活疫苗对于亚洲和非洲经济较落后的发展中地区来说价格太高，限制了它们在发展中国家的使用，而大部分经济发达的城市仍然首选进口灭活疫苗。国产兽用灭活疫苗需要进一步降低成本。 发明内容 本发明要解决的技术问题是克服现有疫苗成本较高等缺陷和不足，提供青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用。 本发明的目的是提供青蒿琥酯(简称(ART))作为活性成分在制备狂犬病疫苗佐剂中的应用。 将ART与狂犬病毒疫苗株混合免疫小鼠，增强灭活狂犬病病毒在小鼠中的免疫应答。并进一步对ART作为疫苗佐剂进行鉴定以及与疫苗混合后疫苗的效果评估，实验结果发现，青蒿琥酯可显著增强狂犬病疫苗的免疫原性，可以作为新型狂犬病疫苗候选佐剂。 在本发明的一实施例中，所述青蒿琥酯作为唯一活性成分。 本发明还提供一种狂犬病疫苗佐剂，所述狂犬病疫苗佐剂包含青蒿琥酯。 在本发明的一实施例中，所述青蒿琥酯作为唯一活性成分。 本发明还提供青蒿琥酯在制备狂犬病疫苗中的应用，所述青蒿琥酯作为佐剂。 本发明还提供一种疫苗组合物，其包含灭活的狂犬病病毒毒株和青蒿琥酯。 在本发明的一实施例中，所述灭活的狂犬病病毒毒株选自CVS-11毒株、rHEP-dG毒株、SAD株、CTN-1株、PV株、FluryLEP株中的至少一种。当然，不限于上述毒株，所有狂犬病病毒毒株均适用于本发明。 在本发明的一实施例中，所述疫苗组合物中狂犬病病毒毒株与青蒿琥酯的用量比为0.9×105～1.1×105FFU：120～130μg。 在本发明的一实施例中，所述疫苗组合物中狂犬病病毒毒株的含量为1.0×105～1.1×105FFU：120～130μg。 在本发明的一实施例中，所述疫苗组合物中青蒿琥酯的用量为5μg/g动物体重。青蒿琥酯的用量是根据小鼠等动物体重确定，以小鼠为例，实际使用浓度为5μg/g小鼠体重，如果每只小鼠体重约为25g，那么，青蒿琥酯的用量约为125μg。 在本发明的一实施例中，还包括二甲基亚砜(DMSO)，也可以采用其他溶剂，DMSO在溶解一些水溶性好的试剂时，溶解效果更好。 在本发明的一实施例中，所述疫苗组合物中青蒿琥酯(g)：二甲基亚砜(mL)＝1：(8～12)，优选为1:10。 在本发明的一实施例中，还包括PBS缓冲液。PBS缓冲液具有生理盐水功效，在稳定蛋白方面更具有优势，pH为7.20-7.40。 在本发明的一实施例中，按体积计，所述疫苗组合物中青蒿琥酯：PBS缓冲液＝1：(90～110)，优选为1：100。 本发明还提供青蒿琥酯在制备狂犬病疫苗中的应用。该狂犬病疫苗用于预防或治疗人或其他动物的狂犬病。 可选地，所述青蒿琥酯作为佐剂。 术语“佐剂”是改变其他药剂功效的药学或免疫学试剂或组合物，在广义上是指自身不提供免疫，但能够增加共同施用的抗原的免疫原性的广谱物质。佐剂可以添加到疫苗中，通过增加免疫应答来增加抗体的数量和持久的保护，从而最大限度地减少注射的外来物质。佐剂也可用于提高疫苗的效力，通过帮助改变对特定类型的免疫系统细胞的免疫反应，例如，根据疫苗的目的，通过激活T细胞而不是分泌抗体的B细胞。因此，佐剂可以有利地调节细胞因子表达/分泌、抗原呈递、免疫应答的类型等。在本发明中，该术语的情形是指作为免疫原和/或其他药物活性化合物的载体或辅助物质的化合物或组合物。 其中上述“抗原”典型地是指可以由免疫系统识别并能够例如通过形成作为适应性免疫应答的一部分的抗体或抗原特异性T-细胞来触发抗原特异性免疫反应的物质。抗原根据性质分为两类：完全抗原和不完全抗原。完全抗原(complete antigen)简称抗原，是一类既有免疫原性，又有免疫反应性的物质，如大多数蛋白质、细菌、病毒、细菌外毒素和肠毒素等。不完全抗原即半抗原(hapten)是只具有免疫反应性，而无免疫原性的物质，如绝大多数多糖(如肺炎球菌的荚膜多糖)、荚膜多糖的水解产物和所有的类脂等。 本发明具有以下有益效果： 本发明首次提出将青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂。 本发明通过实验研究发现青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂能显著增强狂犬病疫苗的免疫原性，可以作为新型狂犬病疫苗候选佐剂。 本发明的疫苗效果研究显示，较低剂量的青蒿琥酯(ART)能促进狂犬病疫苗的免疫效果，可以降低制作疫苗的成本。 本发明的疫苗效果研究显示，青蒿素衍生物青蒿琥酯(ART)作为狂犬病疫苗佐剂能产生更好的保护力。 附图说明 图1显示为一种青蒿素衍生物青蒿琥酯(ART)的化学式。 图2显示为ART对KM小鼠体重影响统计图，同时设立PBS对照组。 图3显示为将ART联合灭活的CVS-11免疫小鼠后外周血抗狂犬病病毒中和抗体水平统计图，同时设立PBS对照组(*P<0.05)。 图4显示为将ART联合灭活的rHEP-dG免疫小鼠后外周血抗狂犬病病毒中和抗体水平统计图，同时设立PBS对照组(*P<0.05)。 图5显示为ART作为疫苗佐剂免疫小鼠14天后CVS-11攻击小鼠的存活率统计图。 具体实施方式 以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。 除非特别说明，以下实施例所用试剂和材料均为市购。 目前，世界卫生组织根据其安全性推荐使用灭活狂犬病疫苗，为了开发更有效和更便宜的灭活疫苗，我们可以采取提高病毒滴度或寻找更好的佐剂等手段。 图1显示为以下实施例所采用的青蒿素衍生物青蒿琥酯(ART)的化学式，青蒿琥酯(ART)的CAS号为88495-63-0，分子式：C19H28O8，分子量：384.42，EINECS号：618-170-5。 以下实施例中所使用的青蒿琥酯购于TCI AmericaPortland公司(东京化成工业株式会社)。 以下实施例中的青蒿素衍生物青蒿琥酯(ART)佐剂，是以灭活的CVS-11或rHEP-dG毒株与ART混合，形成狂犬病病毒疫苗。 具体地：ART首先溶解在DMSO中，然后在体内使用时溶解在PBS中与灭活的CVS-11或rHEP-dG狂犬病病毒混合。按照青蒿琥酯(g)：DMSO(mL)＝1:10的比例，即1g的青蒿琥酯溶解在10mL DMSO中，然后将溶解在DMSO中的青蒿琥酯再溶解到PBS缓冲液中，青蒿琥酯与PBS缓冲液的体积比为1:100，PBS缓冲液即为磷酸盐缓冲液，pH为7.2-7.4。 使用该ART作为佐剂，能够促进狂犬病疫苗诱导产生更高的抗狂犬病病毒中和抗体，且产生更好的保护效果，降低了犬用疫苗的成本。 具体地，该免疫佐剂中，青蒿琥酯是按5μg/g小鼠体重的剂量与1.0×105FFU灭活的CVS-11或rHEP-dG混合，通过右腿腓肠肌肌内注射免疫小鼠后收集外周血，利用中和实验研究抗狂犬病病毒中和抗体效价。 另外，青蒿素衍生物青蒿琥酯(ART)在作为疫苗佐剂或制备疫苗方面的应用，也在本发明的保护范围之内。 青蒿素衍生物青蒿琥酯(ART)首先溶解在DMSO中，然后在体内使用时溶解在PBS中，与灭活的CVS-11或rHEP-dG狂犬病病毒混合后使用。该免疫佐剂青蒿琥酯是按5μg/g的剂量与灭活的CVS-11或rHEP-dG混合，通过右腿腓肠肌肌内注射免疫小鼠后收集血清，采用中和实验检测抗狂犬病病毒中和抗体效价。本发明还对ART副作用、ART佐剂疫苗免疫原性和攻毒保护力等生物学特性进行研究，以评估其作为疫苗佐剂的安全性和效果。结果显示，ART作为新型疫苗佐剂在小鼠体内不会产生显著的副作用，ART作为佐剂能显著增强疫苗的免疫原性，促进疫苗诱导机体产生具有保护效力的抗狂犬病病毒中和抗体，能够更好的保护机体抵抗致死性狂犬病病毒的攻击。 具体研究方法方面，本发明利用免疫学相关技术，研究了在小鼠体内ART作为新型疫苗佐剂的效果评估。 实施例1 新型狂犬病病毒疫苗佐剂ART对小鼠体重影响的研究 将3-4周龄的SPF级昆明(KM)小鼠分为2组，即ART组和PBS对照组，每组包含6只小鼠。ART以5μg/g的剂量通过小鼠右腿腓肠肌肌肉进行注射，对照组注射等量PBS。给药后每天检测小鼠的体重，一共监测15天。将所有小鼠的体重比上各自注射前的体重，研究体重比例变化情况。如图2所示，结果表明，ART不会导致治疗小鼠体重减轻，这与PBS治疗相似。更重要的是，治疗小鼠没有出现异常症状。因此，剂量为5μg/g的ART治疗对小鼠是安全的。 实施例2 青蒿琥酯(ART)对狂犬病疫苗在KM小鼠体内的免疫原性及攻毒保护研究 1、实验方法 分别将CVS-11和rHEP-dG用UV灭活。然后将ART与灭活的CVS-11或rHEP-dG混合，PBS用作佐剂对照。6～7周龄的SPF级昆明系小鼠分为4组，即ART+CVS-11组、PBS+CVS-11组、ART+rHEP-dG组和PBS+rHEP-dG对照组，每组包含6只小鼠。实验组每只小鼠右腿腓肠肌注射1.0×105FFU灭活的狂犬病病毒和ART的混合物，混合物的体积为200μL，混合物中ART的含量为5μg ART/g小鼠体重，青蒿琥酯(g)：二甲基亚砜(mL)＝1：10，按体积计，青蒿琥酯：PBS缓冲液＝1：100，对照组肌注射1.0×105FFU灭活的狂犬病病毒和PBS混合物。在免疫后第7天和第14天采集外周血，并且通过荧光抗体病毒中和(FAVN)试验检测血清抗狂犬病病毒中和抗体水平，抗狂犬病标准血清购自世界卫生组织(WHO)，检测方法严格按照世界动物卫生组织(World Organisation for Animal Health,OIE)的标准操作规程。 2、实验结果 如图3所示，结果表明，用灭活的CVS-11联合ART免疫小鼠产生的狂犬病病毒中和抗体水平显著高于PBS对照组。如图4所示，用灭活的rHEP-dG联合ART免疫小鼠产生的狂犬病病毒中和抗体水平显著高于PBS对照组。 图3和图4的结果表明，ART作为佐剂应用于狂犬病疫苗时，并不局限于某一种特定的灭活狂犬病病毒毒株，而是可以适用于各种灭活的狂犬病病毒毒株。 3、进一步地，为了验证小鼠获得高水平免疫力后是否具有对强毒攻击的保护，在免疫后第14d将活的CVS-11毒株通过肌肉注射感染小鼠，同时设置没有免疫狂犬病疫苗的对照组，连续3周观察小鼠的发病及死亡情况，每天记录存活率。如图5所示，结果表明，用ART作为佐剂的小鼠比用PBS对照的小鼠具有更高的存活率。 上述结果表明，青蒿素衍生物青蒿琥酯(ART)在用作佐剂时增强了灭活狂犬病疫苗的免疫应答。 上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。 Claims (9) 1.青蒿琥酯作为活性成分在制备狂犬病疫苗佐剂中的应用。 2.根据权利要求1所述的狂犬病疫苗佐剂，其特征在于，所述青蒿琥酯作为唯一活性成分。 3.一种狂犬病疫苗佐剂，其特征在于，所述狂犬病疫苗佐剂包含青蒿琥酯。 4.根据权利要求3所述的狂犬病疫苗佐剂，其特征在于，青蒿琥酯作为唯一活性成分。 5.青蒿琥酯在制备狂犬病疫苗中的应用，其特征在于，所述青蒿琥酯作为佐剂。 6.一种疫苗组合物，其特征在于，其包含灭活的狂犬病病毒毒株和青蒿琥酯。 7.根据权利要求6所述的疫苗组合物，其特征在于，所述灭活的狂犬病病毒毒株选自CVS-11毒株、rHEP-dG毒株中的至少一种。 8.根据权利要求6所述的疫苗组合物，其特征在于，所述疫苗组合物中狂犬病病毒毒株与青蒿琥酯的用量比为0.9×105～1.1×105FFU：120～130μg。 9.根据权利要求8所述的疫苗组合物，其特征在于，所述疫苗组合物中狂犬病病毒毒株与青蒿琥酯的用量比为1.0×105～1.1×105FFU：120～130μg。 Patent Citations (4) Publication numberPriority datePublication dateAssigneeTitle CN1758905A *2003-02-122006-04-12乔治敦大学青蒿素在治疗致癌病毒诱导的肿瘤和治疗病毒感染中的应用 WO2010105096A2 *2009-03-112010-09-16University Of MassachusettsModulation of human cytomegalovirus replication by micro-rna 132 (mir132), micro-rna 145 (mir145) and micro-rna 212 (mir212) WO2014124430A1 *2013-02-112014-08-14Emory UniversityNucleotide and nucleoside therapeutic compositions and uses related thereto CN109675027A *2017-10-182019-04-26辽宁成大生物股份有限公司一种狂犬病疫苗佐剂、疫苗组合物及其应用 Family To Family Citations * Cited by examiner, † Cited by third party Non-Patent Citations (2) Title EGERUAN B. 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Dhungyel et al.2014Footrot vaccines and vaccination Marcinkiewicz et al.2018Eliminating factor H-binding activity of Borrelia burgdorferi CspZ combined with virus-like particle conjugation enhances its efficacy as a Lyme disease vaccine CN107073097A2017-08-18双佐剂疫苗组合物、制备和用途 CN107296955B2021-11-23免疫原性支气管炎博德特氏菌组合物 Relyveld et al.1983[3] Preparation of vaccines by the action of glutaraldehyde on toxins, bacteria, viruses, allergens, and cells KR0162074B11998-12-01항원용액중의 보조제로서 수산화아연 또는 수산화철의 용도, 및 이러한 보조제를 함유하는 항원용액 KR19980025002A1998-07-06개량된 불활성 백신 TWI648063B2019-01-21用於免疫以對抗艱難梭菌（Ｃ. difficile）之組合物及方法 CN110507819A2019-11-29青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用 CN100572532C2009-12-23一种鸭瘟疫苗及其专用毒株 Grubhofer1995An adjuvant formulation based on N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine with dimethyldioctadecylammonium chloride and zinc-L-proline complex as synergists Sampath et al.2005An immunogenicity study of a newly introduced purified Vero cell rabies vaccine (Abhayrab) manufactured in India Hosty et al.1959Human antirabies gamma globulin Panse et al.1964Passive immunity in experimental cholera CN110179975B2021-01-08植物油疫苗佐剂及其制备方法和用途 CN101717779B2012-02-01一种适用于炭疽抗毒素和疫苗的融合蛋白 WO2016036503A12016-03-10Antigen compositions and methods of use for treating extraintestinal pathogenic e. coli infections DK2376113T32014-12-08PREPARATIONS AND DOSAGE REGIMES WHICH INCLUDES A CLOSTRIDIUM VACCINE AND levamisole US6905712B22005-06-14Vaccine adjuvants comprising ginseng plant extract and added aluminum salt Priority And Related Applications Priority Applications (1) ApplicationPriority dateFiling dateTitle CN201910745298.1A2019-08-132019-08-13青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用 Applications Claiming Priority (1) ApplicationFiling dateTitle CN201910745298.1A2019-08-13青蒿琥酯作为免疫佐剂在制备狂犬病疫苗中的应用

The invention relates to effects and application of artemisinin and its derivative in inhibition of platelet-derived growth factor receptor A. Particularly, artemisinin and its derivative directly act on a molecular target platelet-derived growth factor receptor A (PDGFR alpha) of a tumor and combines with PDGFR alpha on a cell membrane to inhibit activation of tyrosine kinase at the intracellular domain of PDGFR alpha, inhibit protein stability of PDGFR alpha, and promote a ubiquitin-dependent proteasome mediated degradation process. The artemisinin and its derivative has dose-dependent cell growth inhibition and migration invasion inhibition effects on PDGFR alpha highly expressed tumor cells, and also can inhibit activation of signaling pathways PI3K/AKT and MAPK/ERK. 本发明涉及青蒿素及其衍生物在抑制血小板衍生生长因子受体A中的作用及其应用，具体地，青蒿素及其衍生物直接作用于肿瘤的分子靶标血小板衍生生长因子受体A(PDGFRα)，与细胞膜上的PDGFRα结合，抑制PDGFRRα胞内段的酪氨酸激酶的活化，抑制PDGFRα的蛋白稳定性，促进泛素依赖的蛋白酶体介导的降解过程，对于高表达PDGFRα的肿瘤细胞具有剂量依赖的细胞生长抑制和迁移侵袭抑制作用，也可以抑制信号通路PI3K/AKT和MAPK/ERK的活化。 Effect and the application thereof in suppressing platelet derived growth factor receptor A of arteannuin and derivant thereof Technical field The present invention relates to biomedicine field, particularly, the present invention relates to effect and the application thereof in suppressing platelet derived growth factor receptor A of arteannuin and derivant thereof. Background technology Herba Artemisiae Annuae has another name called Herba Artemisiae annuae (Artemisia anaua L), belongs to Compositae, is the annual herb plant.Arteannuin is the sesquiterpene lactones class antimalarial agent that China pharmacy worker extracted from the Chinese medicine Herba Artemisiae Annuae in early 1970s.In view of traditional quinine class medicine generally runs into the Drug resistance problem, arteannuin and its derivant have replaced them at present, become antimalarial main flow medicine in the world.An endoperoxide bridge is arranged on the macro ring of arteannuin, under the catalysis of ferrous ion or heme, can emit the free radical centered by carbon.Intracellular protein and nucleic acid are dead after by these free radical alkanisations.Plasmodium colonizes in the erythrocyte, contains in a large number the hemes from hemoglobin, thereby arteannuin easy being activated very, and then plasmodium is killed.Arteannuin and its derivant, as dihydroartemisinine, Artemether, arteether and artesunate etc. has been widely used in malaria treatment, and more artemisinin derivative is developed. The antimalarial active of arteannuin and derivant thereof has obtained universally acknowledged, have rapid-action, drug effect is high, less toxic side effect and other advantages.Except being widely used in malaria treatment, artemisinin-based drug also has other multiple pharmacological effect, as anti-schistosome function, arrhythmia, relieving asthma, effects such as antiendotoxin, antiallergic action, anti-lupus erythematosus, immunosuppressant. Along with deepening continuously to arteannuin and derivatives active research thereof, it is found that, this compounds is inhibited and excellent curative to the growth of kinds of tumor cells, yet its concrete action target spot and coherent signal path and mechanism of action are also indeterminate.Therefore this area presses for and understands the effect in tumor always of arteannuin and derivant thereof. Summary of the invention The purpose of this invention is to provide the effect in suppressing platelet derived growth factor receptor A of arteannuin and derivant thereof. Another object of the present invention provides arteannuin and derivant thereof as the application of platelet derived growth factor receptor A inhibitor/antagonist. Another object of the present invention provides the method for external treatment tumor. In a first aspect of the present invention, provide a kind of arteannuin and derivant thereof for the preparation of suppressing to be selected from down the medicine of group disease or the purposes of medicine sensitizer: PDGFR α positive tumor, vascular conditions or fibrous lesions. In another preference, described PDGFR α positive tumor cell is the positive ovarian cancer cell of PDGFR α or PDGFR α masculine liver cancer cell. At second portion of the present invention, provide the purposes of a kind of arteannuin and derivant thereof, for the preparation of inhibitor or the antagonist of platelet derived growth factor receptor A (PDGFR α). In another preference, described platelet derived growth factor receptor A (PDGFR α) comes from the people. In another preference, described inhibitor or antagonist also are used for: (i) activation of inhibition PDGFR α tyrosine kinase; And/or The protein stability that (ii) suppresses PDGFR α; And/or (iii) promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; And/or (vi) suppress the activation of PDGFR α positive tumor cell signal path PI3K/AKT and MAPK/EPK. In another preference, described artemisinin derivative is selected from down group: Artemether, dihydroartemisinine, artesunate, two hydrogen Artemether, arteether etc. In another preference, described artemisinin derivative is dihydroartemisinine. In a third aspect of the present invention, provide a kind of external non-therapeutic ground to suppress the active method of platelet derived growth factor receptor A (PDGFR α), comprise step: with arteannuin and/or artemisinin derivative and cells contacting, thus platelet derived growth factor receptor A (PDGFR α) in inhibition or the antagonism cell. In another preference, described contact is in the presence of arteannuin and/or artemisinin derivative, cultivates described cell. In another preference, described cell comprises tumor cell, preferably comprise PDGFR α positive tumor cell, more preferably comprise the positive ovarian cancer cell of PDGFR α or hepatoma carcinoma cell, as ovarian cancer cell A2780, ovarian cancer cell OVCAR3, hepatoma carcinoma cell Hep3B. In another preference, described method also is used for following at least a kind of application: External non-therapeutic ground suppresses the activation of PDGFR α tyrosine kinase; External non-therapeutic ground suppresses the protein stability of PDGFR α; External non-therapeutic ground promotes the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; External non-therapeutic ground suppresses the activation of signal path PI3K/AKT and MAPK/EPK; With External non-therapeutic suppresses the growth of PDGFR α positive tumor cell. In a fourth aspect of the present invention, a kind of purposes of preparation combination is provided, described preparation combination comprises: (I) contain the preparation of arteannuin and/or artemisinin derivative; With (II) contain the preparation of gemcitabine or carboplatin, The combination of described preparation for the preparation of: (a) pharmaceutical composition or the medicine box of inhibition platelet derived growth factor receptor A; Or (b) prevent and/or treat pharmaceutical composition or the medicine box of tumor. In another preference, described preparation comprises: tablet, capsule, suppository, injection. In another preference, described tumor is PDGFR α positive tumor. In a fifth aspect of the present invention, a kind of method of the PDGFR of preventing and/or treating alpha associated disorders is provided, wherein said disease is relevant with expression excessively or the hyperactivity of PDGFR α, described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs, or use the pharmaceutical composition that contains arteannuin and/or artemisinin derivative. In another preference, described PDGFR alpha associated disorders comprises tumor (PDGFR α positive tumor), as ovarian cancer or hepatocarcinoma. In another preference, PDGFR α positive tumor is the positive ovarian cancer of PDGFR α or PDGFR α masculine liver cancer. In another preference, described using comprises: in the tumor, intravenous injection, oral tablet, lumbar injection etc. In another preference, described object is mammal. In a sixth aspect of the present invention, a kind of method is provided, described method is used in the body or growth, migration and the invasion and attack of the activation of vitro inhibition PDGFR α tyrosine kinase, the protein stability of inhibition PDGFR α, the degraded that promotes the proteasome mediation that PDGFR α ubiquitin relies on, the activation that suppresses signal path PI3K/AKT and MAPK/EPK and/or inhibition PDGFR α positive tumor cell, and described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs. In a seventh aspect of the present invention, a kind of test kit for the ovarian cancer pathological grading is provided, described test kit comprises the reagent that detects in vitro tissue PDGFR alpha expression amount. In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus constitute new or optimized technical scheme.As space is limited, this tired stating no longer one by one. Description of drawings Following accompanying drawing is used for explanation specific embodiments of the present invention, and is not used in the scope of the invention that restriction is defined by claims. Fig. 1 shows that PDGFR α can be used as the direct action target of arteannuin and derivant thereof; Wherein, Fig. 1 a is the vitro kinase experiment, the result shows that arteannuin and derivant arteannuin (ART) thereof, dihydroartemisinine (DHA), artesunate (ARM), Artemether (ARS) can suppress the activity of PDGFR α born of the same parents the inner (aa550-end) tyrosine kinase; Fig. 1 b is surface plasma resonance (SPR) experiment, the result shows, dihydroarteannuin (DHA) can mutually combine with PDGFR α bag outer end (aa24-524) albumen, the positive contrast of Suo Lafeini (Sorafenib), known its can be in conjunction with the activity that suppresses PDGFR α; Fig. 1 c is the experiment of biotin-avidin affinity purification, and the result shows that the DHA of labelling biotin can mutually combine with PDGFR α in the A2780 cell, thereby is come out by affinity purification; Fig. 1 d is that area of computer aided is built with pattern and oppositely butt joint experiment (Computer Assisted Homology modeling and docking), the result shows, dihydroarteannuin (DHA) can be fitted to PDGFR α bag outer end PDGF binding site, simultaneously also can be fitted to it and wrap inner ATP-binding site, block its kinase activator; Fig. 1 e-Fig. 1 f shows that dihydroarteannuin can effectively suppress activity and the protein expression of PDGFR α among PDGFR α positive cell A2780 and the OVCAR3, and little to phosphorylation and the protein level influence of PDGFR β; Fig. 1 g-Fig. 1 i shows that dihydroarteannuin can accelerate the protein degradation of PDGFR α, and this process is the proteasome mediation that ubiquitin relies on. Fig. 2 has shown that arteannuin and derivant thereof have growth and the transfer ability of the ovarian cancer cell that suppresses the PDGFRa positive; Wherein, Fig. 2 a shows, after giving medicine irritation 48h, arteannuin and derivant thereof do not have obvious growth to people's normal ovarian epithelial cell IOSE144 and suppress ability, less for the SK-OV3 cytotoxicity, but A2780 and OVCAR3 for the PDGFRa positive have the ability that suppresses more by force, and wherein the effect of arteannuin and dihydroartemisinine is the most remarkable; Fig. 2 b is the cell migration experiment, the result shows, the low concentration short time is handled (12h) ovarian cancer cell, and arteannuin and derivant thereof can suppress the transfer ability of PDGFRa positive cell A2780 effectively, and do not have obvious inhibition ability for the cell SK-OV3 of PDGFRa feminine gender; Fig. 2 c has shown that the dihydroarteannuin of variable concentrations is to the cyto-inhibition of 5 kinds of cell lines (IOSE144, A2780, OVCAR3, OVCAR5, SK-OV3); Fig. 2 d has shown that the dihydroarteannuin of variable concentrations is to the influence of the cell migration of 3 kinds of cell lines (A2780, OVCAR3, SK-OV3); Fig. 2 e has shown that the dihydroarteannuin of variable concentrations is to the influence of the cell invasion of 2 kinds of cell lines (A2780, OVCAR3). Fig. 3 shows that arteannuin and derivant thereof have growth and the transfer ability of the hepatoma carcinoma cell that suppresses the PDGFRa positive; Wherein, Fig. 3 a shows, in the normal hepatic cell line of people (7702 and LO2) and hepatoma cell line (Hep3B, HepG2,7721,7404, LM3,97L, 97M, Huh-7), has only Hep3B high expressed PDGFRa; Fig. 3 b shows that low concentration dihydroarteannuin (3 μ M) (12h) is in short-term handled the Hep3B cell, can significantly suppress its migration and invasive ability; Fig. 3 c is the cell growth experiment, and the result shows that the Hep3B cell of the PDGFRa positive is more responsive for dihydroarteannuin, and other cells are lower for the dihydroarteannuin sensitivity. Fig. 4 shows that dihydroarteannuin can suppress growth and the transfer of mice original position ovarian cancer model; Wherein, Fig. 4 a demonstration dihydroarteannuin is treated mice, and the malignant progression that suppresses ovarian cancer effectively can be arranged; Fig. 4 b shows, compares with matched group, and the situation of the lung cancer metastasis of dihydroarteannuin treatment group mice reduces greatly, and along with the increase of dose, the treatment situation is more good; The two blue and green artemisin of Fig. 4 c demonstration variable concentrations is treated mice, and the situation that send out in the tumor abdominal cavity (lungs, liver, intestinal) obtains obvious suppression; Fig. 4 d is immunohistochemical experiment, and the result shows that its modality process (EMT) of cell that dihydroarteannuin is handled is inhibited; Fig. 4 e is immunoblot experiment, and the result shows that dihydroarteannuin can effectively suppress expression and the phosphorylation of PDGFRa, and downstream signal path PI3K/AKT and MAPK-ERK are suppressed. Fig. 5 has shown the sensitization of arteannuin and derivant and a line chemotherapeutic; Wherein, to be HepG2 and Hep3B cell handle experiment with arteannuin or dihydroarteannuin individual processing or associating gemcitabine to Fig. 1 a, the result shows, gemcitabine acts on HepG2 and Hep3B cell separately, has certain apoptosis facilitation, arteannuin associating gemcitabine all has the effect of remarkable apoptosis facilitation to HepG2 and Hep3B cell, and two medicine association lists reveal synergism; Fig. 5 b shows, DHA and CBP drug combination had the potentiation facilitation to the OVCAR-3 apoptosis after 24 hours, unite the apoptosis rate that apoptosis rate after 500 μ M CBP (1155%) effect is significantly higher than independent usefulness 1 μ M DHA (266%) or 500 μ M CBP (528%) with the DHA of 1 μ M, and surpassed the apoptosis additive effect of two kinds of chemical compounds, be a kind of potentiation, the A2780 cell has shown additive effect to therapeutic alliance; Fig. 5 c shows, when with the DHA coupling, CBP has significantly suppressed the vigor of ovarian cancer cell, when cellular exposure during in 1 μ M CBP and 1 μ M DHA, the survival rate of A2780 cell has just reduced by 69%, OVCAR-3 cell survival and has then reduced 72%, by contrast, it is more insensitive to treatment that the IOSE144 cell then seems, survival rate had only reduced about 28% when two kinds of medicines all share with 1 μ M; Fig. 5 d is hepatoma carcinoma cell Hep3B and the experiment of HepG2 transplanted tumor in nude mice, the result shows, all has antitumor action in arteannuin and the dihydroarteannuin list medicine body, the cancer suppressing action of dihydroarteannuin is better than arteannuin, arteannuin and dihydroarteannuin all can increase the cancer suppressing action of gemcitabine, associating gemcitabine therapeutic effect is better than single medicine, and especially dihydroarteannuin shows significant chemotherapy sensitization; Fig. 5 e is in ovarian cancer cell A2780 and the experiment of OVCAR3 transplanted tumor in nude mice, the result shows, DHA (dosage be 10 with 25mg/kg) causes A2780 heteroplastic transplantation tumor 24% and 41% growth inhibited (comparing with the matched group of giving normal saline) (P＜0.05) respectively, and OVCAR-3 model 14% and 37% tumor growth suppress (P＜0.05); Be suppressed 56% (A2780) and 46% (OVCAR-3) only for the treatment group tumor growth of single CBP, drug combination (25mg/kgDHA) group then all produces 70% tumor growth and suppresses (P＜0.05) in A2780 and OVCAR-3 animal tumor. Fig. 6 shows the expression of PDGFR α and the relation of ovarian cancer pathology rank and transfering state; Wherein, Fig. 6 a shows, compares with the low level ovarian cancer, and in the high-level ovarian cancer case, the expression of tumor cell and Interstitial cell PDGFR α on every side significantly increases; Fig. 6 b is PDGFR α coloration result, and the result shows that in high-level ovarian cancer case, the expression of PDGFR α significantly increases. The specific embodiment The inventor is through extensive and deep research, find the purposes of arteannuin and derivant thereof, inhibitor or antagonist for the preparation of platelet derived growth factor receptor A (PDGFR α), in addition, arteannuin and derivant thereof can also suppress the activation of PDGFR α tyrosine kinase, the protein stability that suppresses PDGFR α, promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on, suppress the activation of signal path PI3K/AKT and MAPK/EPK, suppress the growth of PDGFR α positive tumor cell, migration and invasion and attack, the sensitizer that can also be used for the treatment of PDGFR α positive tumor.Finished the present invention on this basis. Term Ovarian cancer Ovarian cancer is grave danger of global WomanHealth.In the U.S., ovarian cancer has become women's cancer of the 4th lethal.Though diagnosed 5 annual survival rates of sending out ovarian cancer patients early stage in disease can reach 80%-90%, be diagnosed as the ovarian cancer patients survival rate in late period but only less than 25%, in addition, most of ovarian cancer patients are that ovarian cancer is to late period diagnosing out.The mortality rate many decades in the past of ovarian cancer patients does not have to take place too big change at present, therefore in oncotherapy and prognosis, seek effective therapeutic scheme, strengthening tumor cell is research worker and clinical practice personnel target for a long time to the sensitivity of chemotherapeutics and the drug resistance that overcomes tumor. Hepatocarcinoma Hepatocarcinoma is one of modal malignant tumor, and China is the district occurred frequently of hepatocarcinoma, and its sickness rate has risen to second of malignant tumor.Because the early hepatocarcinoma symptom is not obvious, during therefore the most patient of making a definite diagnosis has been in, late period.The first-selection treatment of primary hepatocarcinoma is excision, and along with the raising for the treatment of technology, 5 years survival rates of postoperative have been increased to 50%-60% at present, but because an excision application diameter is no more than 5cm, the cancer number is no more than 3 small liver cancer.In addition, whether be fit to blood vessel enrichment degree and the individual patient difference that direct excision also depends on the cancer position, these restrictions cause the hepatocarcinoma about 90% to be not suitable for operative treatment.Even can excise hepatocarcinoma, it is that main comprehensive therapy also is necessary that postoperative implements to get involved chemotherapy.Yet at present, rarely have medicine can effectively suppress the growth of hepatocarcinoma and further deterioration, hepatocarcinoma is low to the response rate of traditional chemotherapeutics, the toxic and side effects highly significant, so the slow development of chemotherapy is the main cause of restriction antitumor drug clinical practice.Therefore for chemotherapy of tumors, press for and seek better chemical-therapy synergistic agent and better therapeutic scheme, thereby improve tumor to the sensitivity of chemotherapy, reduce it to the drug resistance of chemotherapy. Platelet derived growth factor and receptor thereof Platelet derived growth factor (Platelet derived growth factor, PDGF) be that a class can be synthesized the somatomedin of justacrine in the extracellular matrix, have cell division, propagation, the migration of multiple tissues such as promoting fibrocyte, smooth muscle cell, the ability of sticking of increase cell, in people's fetal development and normal physiological activity, bringing into play important effect.PDGF activates platelet derived growth factor receptor (PDGFR) with autocrine and paracrine form and plays a role. PDGFR comprises tyrosine kinase receptor PDGFR α and the PDGFR β of two kinds of similar, and wherein the abnormal activation of PDGFR α and multiple disease are closely related, and particularly, the PDGFR relevant disease can be divided three classes: 1. tumor Because PDGFR (particularly PDGFR α) is present in the kinds of tumors, it may be as a new treatment target spot.The generation that has been found that kinds of tumors is relevant with the excessive activation of PDGFR (particularly PDGFR α), as ovarian cancer, glioma, carcinoma of prostate; In addition, in some tumors, exist the sudden change of PDGFR to activate, as the chronic myeloid leukemia of dermatofibrosarcoma protuberans, gastrointestinal stromal tumor, Bcr-Abl feminine gender, hypereosinophilic syndrome etc. 2. vascular conditions Discover, PDGF and PDGFR etc. exist overexpression and excessive activation phenomenon in multiple vascular conditions, as atherosclerosis and restenosis, pulmonary hypertension, retinal vasculopathy etc., the symptom of above-mentioned disease can be effectively alleviated in the activation that suppresses the PDGFR signal path, increases its therapeutic effect. 3. fibrous lesions The PDGF-PDGFR signal path plays an important role in multiple fibrous lesions process, as pulmonary fibrosis, hepatic fibrosis and liver cirrhosis, skin fiber hypertrophy (scleroderma), renal fibrosis, myocardial fibrosis etc., wherein the propagation of the Interstitial cell of PDGF-PDGFR signal path mediation is the common trait of this chronic inflammatory disease. In the above-mentioned three class diseases, the PDGF-PDGFR signal path plays an important role, and wherein PDGFR β has mainly mediated the vascular system pathological changes, and PDGFR α has mainly mediated the pathological changes of Interstitial cell and fibroblast driving.(Johanna Andrae, et al.Role of platelet-derived growth factors in physiology and medicine, Genes Dev.2008; 22:1276-1312; Zhang Xiuhua etc., platelet derived growth factor receptor and tumor. life sciences, Vol.18, No.3 Jun., 2006) Arteannuin and derivant thereof Artemisinine is the sesquiterpene lactones medicine that peroxy-radical is arranged that extracts from the Chinese medicine Artemisia annua L., because arteannuin is complicated macromole, chemical total man worker is difficult to synthetic, therefore mainly is biosynthesis and artificial the extraction at present. The biosynthesis of sesquiterpenoidss such as arteannuin carries out in Cytoplasm, approach belongs to Plant Isoprenoid Metabolic Pathway, can be divided into three goes on foot greatly: form FPP by acetic acid, synthetic sesquiterpene, formation arteannuin again lactonizes, be specially: FPP → 4,11-diene sesquiterpene → artelinic acid → dihydroartemisinic acid → dioxy artelinic acid peroxide → arteannuin. The chemical formula of the artemisinin derivative that arteannuin and this area are several frequently seen is as follows: The application of arteannuin and derivant thereof The invention provides arteannuin and derivant thereof for the preparation of the inhibitor of platelet derived growth factor receptor A (PDGFR α) or the purposes of antagonist. In another preference, arteannuin and derivant thereof also are used for: (i) activation of inhibition PDGFR α tyrosine kinase; And/or The protein stability that (ii) suppresses PDGFR α; And/or (iii) promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; And/or (vi) suppress the activation of PDGFR α positive tumor cell signal path PI3K/AKT and MAPK/EPK; And/or (v) suppress growth, migration and the invasion and attack of PDGFR α positive tumor cell; And/or (vi) preparation suppresses medicine or the sensitizer of PDGFR α positive tumor; And/or (vii) medicine or the sensitizer of vascular conditions and fibrous lesions treated in preparation. In a preference of the present invention, provide a kind of external non-therapeutic ground to suppress the active method of platelet derived growth factor receptor A (PDGFR α), comprise step: with arteannuin and/or artemisinin derivative and cells contacting, thus platelet derived growth factor receptor A (PDGFR α) in inhibition or the antagonism cell. Described contact is in the presence of arteannuin and/or artemisinin derivative, cultivates described cell.Cell comprises tumor cell, preferably comprises PDGFR α positive tumor cell, more preferably comprises the positive ovarian cancer cell of PDGFR α or hepatoma carcinoma cell, as ovarian cancer cell A2780, ovarian cancer cell OVCAR3, hepatoma carcinoma cell Hep3B.In another preference, described method also is used for following at least a kind of application: external non-therapeutic ground suppresses the activation of PDGFR α tyrosine kinase; External non-therapeutic ground suppresses the protein stability of PDGFR α; External non-therapeutic ground promotes the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; External non-therapeutic ground suppresses the activation of signal path PI3K/AKT and MAPK/EPK; Growth with external non-therapeutic inhibition PDGFR α positive tumor cell. The present invention also provides a kind of method of the PDGFR of preventing and/or treating alpha associated disorders, wherein said disease is relevant with expression excessively or the hyperactivity of PDGFR α, described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs, or use the pharmaceutical composition that contains arteannuin and/or artemisinin derivative.The PDGFR alpha associated disorders comprises tumor (PDGFR α positive tumor), as ovarian cancer or hepatocarcinoma.In another preference, described using comprises: in the tumor, intravenous injection, oral tablet, lumbar injection etc.Described object is mammal (as the people). Medicament composition and application thereof The present invention also provides medicament composition, and in a preference, described preparation combination comprises: (I) contain the preparation of arteannuin and/or artemisinin derivative; (II) contain the preparation of gemcitabine or carboplatin, The combination of described preparation for the preparation of: (a) suppress pharmaceutical composition or the medicine box of platelet derived growth factor receptor A; Or (b) prevent and/or treat pharmaceutical composition or the medicine box of tumor. Described preparation comprises tablet, capsule, suppository, injection; Described pharmaceutical preparation is used for prevention and treatment PDGFR α positive tumor. Medicament composition of the present invention can be solid or liquid.But solid preparation comprises powder, tablet, pill, capsule, cachet, suppository and dispersible granule, and solid carrier can be one or more materials, and they can be used as diluent, correctives, adhesive, antiseptic, tablet disintegrant or coating material.In powder, carrier is the solid of fine branch, and the active constituent of chemical compound of the present invention and fine branch is present in the mixture.In tablet, this chemical compound mixes with proper proportion with required bonding carrier, and is pressed into required shape and size.Preferentially contain 5 to 70% reactive compounds in powder and the tablet, suitable carrier is magnesium carbonate, magnesium stearate, Talcum, sugar, lactose, pectin, dextrin, starch, gelatin, carboxymethyl cellulose, sodium carboxymethyl cellulose, low melting point is cured and cocoa butter etc.Equally, cachet or lozenge, tablet, powder, capsule, pill, cachet and lozenge can be the solid dosage formss that is suitable for oral administration.In order to prepare suppository, can be with the mixture melt of the cured butter oil acid glyceride of low melting point or cocoa butter, and by stirring this reactive compound component is evenly dispersed in wherein, the homogeneous mixture of fusing is poured into allowed its cooling in the sizeable mould then, and solidify thus.The preparation of solution form comprises solution, suspending agent and Emulsion, for example water or aqueous propylene glycol solution.For non-intestinal injecting fluid preparation, can in moisture polyglycol solution, prepare.Being suitable for oral aqueous solution can be by being dissolved in the water active constituent, and adds suitable coloring agent, correctives, emulsifying agent and thickening agent preparation as required.Be suitable for oral aqueous suspensions and can be scattered in by the active constituent with fine branch in the moisture stickum and prepare, as natural or rubber polymer, resin, methylcellulose, Carboxymethyl cellulose sodium and other suspensoids of knowing.These liquid forms comprise solution, suspensoid and Emulsion, and except active constituent, these preparations can contain coloring agent, correctives, stabilizing agent, buffer agent, synthetic or natural sweetener, dispersant, thickening agent and cosolvent etc.This pharmaceutical preparation preferentially is unit dosage forms, in this dosage form, this preparation is subdivided into the unit dose that contains an amount of active constituent, and this unit dose can be the preparation of packing, this packing contains a certain amount of preparation, as tablet, capsule and the powder in bottle or capsule of packing.This unit dosage forms can also be capsule, tablet, cachet or lozenge itself, or can there be any of these powder of the right quantity in the packaged form in it. The amount of active constituent can change according to the effectiveness of concrete application and active constituent in the unit dose formulations, treat for non-human mammal (as mice), can be with 0.01mg to about 0.5g, preferably 0.1 to about 3mg capsule is administered three times every day, and said composition can also contain other compatible therapeutic agents in case of necessity.Treatment for the people, can be according to patient's needs, changed by the chemical compound of sanatory seriousness and use, preferably, beginning is with the smaller dose treatment less than this chemical compound optimal dose, after this, increasing this dosage reaches optimum efficiency in a small amount, for the purpose of making things convenient for, total daily dose can be subdivided into again gradation administration in a day if desired.In a preference of the present invention, the dosage of administration of human is approximately between the 0.1/kg-0.5g/kg. Major advantage of the present invention: (1) arteannuin and derivant thereof can be for the preparation of inhibitor or the antagonisies of platelet derived growth factor receptor A (PDGFR α); (2) arteannuin and derivant thereof can suppress growth, migration and the invasion and attack of the activation of PDGFR α tyrosine kinase, the protein stability of PDGFR α, the activation that suppresses signal path PI3K/AKT and MAPK/EPK, inhibition PDGFR α positive tumor cell; (3) arteannuin and derivant thereof can promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; (4) arteannuin and derivant thereof can be used for the sensitizer of PDGFR α positive tumor treatment. Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer. Experimental technique and material 1. surface plasma resonance Surface plasma resonance (SPR) is a kind of optical phenomena, can be used to real-time tracking interaction between biomolecule under native state.In the experiment, with recombined human PDGFR α born of the same parents outer ends (Met1-Glu524) albumen, be bonded in biosensor surface, with the dihydroarteannuin solution of variable concentrations with have interactional chemical compound Suo Lafeini and SU11248 solution to inject and the biosensor surface of flowing through with PDGFR α always.Combination between biomolecule causes the increase of biosensor surface quality, causes refractive index to strengthen in same ratio, and the variation of reacting between biomolecule namely is observed, and this reaction is weighed with reacton (RU). 2. cell culture The non-tumorigenic epithelial cell IOSE-144 of people's ovary immortalization and ovarian cancer cell (A2780, OVCAR-3, SK-OV3, OVCAR-5) available from U.S. ATCC (American Type Culture Collection, Manassas, VA).All cells requires (RPMI-1640 or DMEM culture fluid add 10% hyclone (GIBCO), 100IU/ml penicillin, 100IU/ml streptomycin) to cultivate in 37 ℃ of incubators of 5%CO2 according to the cultivation of ATCC. 3. cell growth inhibition test Cell growth and propagation inhibition test are by the CCK-8 measuring.Cell with the density cover plant in 3 * 103 every holes of cell in 96 orifice plates.After 12～18 hours, after handling 48 hours with the artemisine compounds of finite concentration gradient (0,1,5,10,25 μ M), add the cells growth activity evaluation after 10 μ L CCK-8 solution carry out drug treating in the every hole of 96 orifice plates, experiment is last by microplate reader (SpectraMax190microplate reader; Molecular Devices, the USA) light absorption (OD value) at measurement 450nm place (every experimental concentration arranges three multiple holes at least, tests triplicate at least at every turn). 4. cell migration, invasion and attack experiment The migration of ovarian cancer cell and invasive ability are by the transwell measuring, the low concentration artemisinin derivative short time (12h) is handled ovarian cancer cell, cell (5 * 104) with similar number is resuspended in the culture medium of serum-free then, plantation is in transwell (when measuring the invasive ability of cell, transwell spreads matrigel in the upper strata) upper strata culturing room.With 10% hyclone culture medium as chemical chemoattractant.Approximately behind the 8-12h, Qian Yi cell is not wiped with cotton swab, the cell of migration is fixed dyeing, and carry out statistical analysis. 5.SDS-polyacrylamide gel electrophoresis and protein immunoblot analysis DHA with variable concentrations handled cultured cell 24 hours, and collecting cell is also used cell pyrolysis liquid (RIPA buffer #9806, Cell Signaling) cracking.Change in 4 ℃ of centrifugal 15min with 13,000 afterwards, get supernatant and carry out follow-up analyzing and testing.Protein concentration is by test kit (the protein assay kit of Bio-Rad company; Hercules, CA) quantitative Tot Prot.Equal protein is carried out the separation of SDS-PAGE gel electrophoresis on every hole afterwards.Electrophoresis finishes, with the electrophoresis protein band be transferred to the pvdf membrane that methanol activated (Millipore, Bedford, MA) on.After the commentaries on classics film finishes, film seals with 5% skim milk (PBS dissolving), add primary antibodie and two anti-hatching (the BSA configuration with 5%) then, change with albumen development test kit (ECL plus system, Amersham Pharmacia Biotech) development by the protein band differential expression afterwards and analyze. 6. mice original position ovarian cancer heteroplastic transplantation tumor model body internal therapy experiment The female BALB/c (nu/nu) in four to six ages in week and raises in SPF level Animal House in accordance with regulations available from Shanghai Slac Experimental Animal Co., Ltd. (Shanghai Experimental Animal Center).All zoopery operations all obtain the approval of animal Ethics Committee of nutrition institute of the Chinese Academy of Sciences.。General steps is as follows: with the A2780 cell of luciferase labelling, be resuspended in RPMI 1640 culture medium of serum-free.Lumbar injection equivalent cell (～3 * 106 cells/0.2ml) is in the inside, abdominal cavity of mice then.Utilize IVIS Lumina bioluminescence system to monitor the growth of tumor situation in real time, and the body weight of record mice.Inject after 5 days, during according to the fluorescence intensity of tumor mice is divided into treatment group and matched group (every group of 8 mouse).Treatment group medicine DHA be dissolved in Oleum Ricini, ethanol and normal saline mixture (Cremophor EL: Ethanol: Saline=5: 5: 90, v/v/v) in, all medicines all pass through intraperitoneal administration.The dosage of DHA is 10 or the 25mg/kg body weight, and administration frequency is administration every day, rests weekly two days.The matched group injecting normal saline.In therapeutic process, utilize the living imaging system to monitor the growth of tumor situation in real time.After treatment finished, the careful taking-up moved abdominal tumor (removing fat and connective tissue), with PIPA solution (the 100mg tissue adds 1ml RIPA) homogenate.The tumor tissues homogenate liquid that obtains is analyzed requirement of experiment by above-mentioned protein immunoblot and is handled, and carries out protein immunoblot afterwards and detects; Or tumor tissues fixed and immunostaining. Embodiment 1 Arteannuin and derivant thereof suppress its phosphorylation activity directly in conjunction with PDGFR α, promote its degraded Utilize the experiment of PDGFR α vitro kinase, surface plasma resonance, area of computer aided are built with pattern and oppositely advanced persons such as equimolecular pharmacology and biophysics are tested in butt joint biology techniques and means, confirm that artemisinin derivatives can be directly in conjunction with PDGFR α, the receptor tyrosine kinase activity that suppresses PDGFR α, discover that simultaneously dihydroarteannuin can suppress the protein stability of PDGFR α in the PDGFR α positive cell, promote the degradation process that its ubiquitin relies on. Fig. 1 shows that arteannuin and derivant thereof can be directly in conjunction with PDGFR α, and PDGFR α is as the direct target of arteannuin and derivant effect thereof, and can suppress its phosphorylation activity, promotes its degraded.Vitro kinase experiment shows arteannuin and derivant thereof: arteannuin (ART), dihydroartemisinine (DHA), artesunate (ARM), Artemether (ARS) can suppress the activity of born of the same parents the inner (aa550-end) tyrosine kinase of PDGFR α (Fig. 1 a).Surface plasma resonance (SPR) experiment shows that dihydroarteannuin (DHA) can mutually combine with PDGFR α bag outer end (aa24-524) albumen, and Suo Lafeini (Sorafenib) is as positive control, known can be in conjunction with the activity (Fig. 1 b) that suppresses PDGFR α.The DHA of biotin-avidin affinity purification experiment show tags biotin can mutually combine with PDGFR α in the A2780 cell, thereby is come out (Fig. 1 c) by affinity purification.Area of computer aided is built and is oppositely docked experiment (Computer Assisted Homology modeling and docking) experiment and shows that dihydroarteannuin (DHA) can be fitted to PDGFR α bag outer end PDGF binding site with pattern, simultaneously also can be fitted to it and wrap inner ATP-binding site, block its kinase activator (Fig. 1 d).Fig. 1 e-Fig. 1 f has shown that dihydroarteannuin can effectively suppress activity and the protein expression of PDGFR α among PDGFR α positive cell A2780 and the OVCAR3, and little to phosphorylation and the protein level influence of PDGFR β.Fig. 1 g-Fig. 1 i experiment shows that dihydroarteannuin can accelerate the protein degradation of PDGFR α, and this process is the proteasome mediation that ubiquitin relies on. Embodiment 2 Arteannuin and derivant thereof suppress growth and the transfer of the tumor cell of the PDGFR α positive Utilize nude mice original position ovarian cancer heteroplastic transplantation tumor model, research dihydroarteannuin ovarian cancer resistance activity in animal body.The Proliferation of Human Ovarian Cell A2780 of luciferase labelling is injected into the abdominal cavity of Balb-c nude mice, sets up the former bit model of nude mice ovarian cancer, give the dihydroarteannuin treatment then: matched group (normal saline), low dose group (DHA 10mg/kg).High dose group (DHA 25mg/kg) is treated weekly five times, rests two days, gives for 6 treatment times in week altogether.Utilize growth and the transfer case of living imaging systematic observation mouse ovarian cancer during this time. A series of researchs by cellular level and animal level, show that artemisinin derivatives can suppress growth, migration and the invasion and attack (ovarian cancer and hepatocarcinoma) of the tumor cell of the PDGFR α positive effectively, and less for the inhibition ability of the growth of tumour cell of PDGF-B expression R α not and transfer ability.Concrete outcome is as follows: Fig. 2 shows that arteannuin and derivant thereof suppress growth and the transfer ability of the ovarian cancer cell of the PDGFRa positive.Particularly, shown in Fig. 1 e and Fig. 2 a, PDGFRa is at ovary normal cell epithelial cell IOSE144, and ovarian cancer cell SK-OV3 does not have among the OVCAR5 and expresses high expressed in ovarian cancer cell A2780 and OVCAR3; After giving medicine irritation 48h, the arteannuin of variable concentrations and derivant people normal ovarian epithelial cell IOSE144 thereof do not have obvious growth and suppress ability, less for the SK-OV3 cytotoxicity, but A2780 and OVCAR3 for the PDGFRa positive have the ability that suppresses more by force, and wherein the effect of arteannuin and dihydroartemisinine is the most remarkable.The cell migration experiment show lower concentration short time is handled (12h) ovarian cancer cell, arteannuin and derivant thereof can suppress the transfer ability of PDGFRa positive cell A2780 effectively, and do not have obvious inhibition ability (Fig. 2 b) for the cell SK-OV3 of PDGFRa feminine gender.Dihydroarteannuin suppresses the most obviously (Fig. 2 c-Fig. 2 e) for growth and the migration invasive ability of PDGFRa positive cells in the artemisinin derivative. Also obtained similar result in the hepatoma carcinoma cell, Fig. 3 shows that arteannuin and derivant thereof can suppress growth and the transfer ability of the hepatoma carcinoma cell of the PDGFRa positive.Particularly, numerous people's normal liver cells (7702 and LO2) and hepatoma cell line (Hep3B, HepG2,7721,7404, LM3,97L, 97M, Huh-7) in, (Fig. 3 is a) for Hep3B high expressed PDGFRa; Low concentration dihydroarteannuin (3 μ M) (12h) processing Hep3B cell in short-term can significantly suppress its migration and invasive ability (Fig. 3 b); The cell growth experiment shows that simultaneously the Hep3B cell of the PDGFRa positive is more responsive for dihydroarteannuin, and express on other PDGFRa ground and the hepatoma carcinoma cell of not having an expression for dihydroarteannuin sensitivity lower (Fig. 3 c). Zoopery assessment result (Fig. 4) shows, dihydroarteannuin has an inhibition ability for the growth of PDGFRa positive cell A2780 and transfer.Particularly, (Fig. 4 a) after two blue and green artemisins are treated the malignant progression that suppresses ovarian cancer effectively; Compare with the contrast group, the situation for shifting that gives dihydroarteannuin treatment group mice reduces greatly, and the situation that send out in the tumor abdominal cavity also obtains obvious suppression (Fig. 4 b, Fig. 4 c); SABC and immunoblot experiment show simultaneously, dihydroarteannuin can suppress expression and the phosphorylation of PDGFRa effectively, downstream signal path PI3K/AKT and MAPK-ERK are suppressed, matter modality process (EMT) be inhibited (Fig. 4 d, Fig. 4 e) between epithelium. Embodiment 3 The sensitization of arteannuin and derivant thereof and a line chemotherapeutic The external independent application of arteannuin and dihydroarteannuin all can significantly suppress growth, propagation and the migration invasive ability of ovarian cancer and hepatoma carcinoma cell.Whether present embodiment has the chemotherapy sensitizing effect to it is detected, arteannuin and dihydroarteannuin are united gemcitabine respectively and are acted on human liver cancer cell HepG2 and Hep3B, and the associating carboplatin acts on the inhibitory action of ovarian cancer A2780 and OVCAR3 observation drug combination cell growth.Result (Fig. 5) is as follows: HepG2, the Hep3B cell is with 10 μ mol/L arteannuin or dihydroarteannuin individual processing or unite 10 μ g/L gemcitabines processing 48 hours, carries out the apoptosis detection by quantitative as stated above.Particularly, gemcitabine list medicine acts on HepG2, and the Hep3B cell demonstrates certain apoptosis facilitation, and (Fig. 5 a), arteannuin associating gemcitabine is to HepG2, the Hep3B cell has the effect of remarkable apoptosis facilitation, and two medicine association lists reveal synergism, and (Fig. 5 a).DHA and CBP drug combination had potentiation facilitation (Fig. 5 b) to the OVCAR-3 apoptosis after 24 hours; Unite the apoptosis rate that apoptosis rate after 500 μ M CBP (1155%) effect is significantly higher than independent usefulness 1 μ M DHA (266%) or 500 μ M CBP (528%) with 1 μ M DHA, and surpassed the apoptosis additive effect of two kinds of chemical compounds, what demonstrate is a kind of potential potentiation.Yet what the A2780 cell showed therapeutic alliance is additive effect rather than synergy.This may be because their causes lower to the sensitivity of DHA chemical compound (Fig. 5 b, P＜0.05).The effect of longer time may produce better effect.Aspect the inhibition of cell proliferation vigor, when with the DHA coupling, CBP has suppressed the vigor of ovarian cancer cell very significantly.In fact, when cellular exposure during in the DHA of 1 μ M CBP and 1 μ M, the survival rate of A2780 cell has reduced by 69%, OVCAR-3 cell survival and has then reduced 72%.By contrast, it is more insensitive to treatment that the IOSE144 cell then seems, survival rate had only reduced about 28% (Fig. 5 c) when two kinds of medicines all share with 1 μ M. Cell experiment proof arteannuin and dihydroarteannuin all demonstrate for gemcitabine external, and carboplatin is handled killing and wounding and apoptosis-induced sensitization of cancerous cell.In addition, the transplanted tumor in nude mice experiment proves that further they are effective too for the chemotherapy of in-vivo tumour.Particularly, show in hepatoma carcinoma cell Hep3B and the experiment of HepG2 transplanted tumor in nude mice, all to have antitumor action in arteannuin and the dihydroarteannuin list medicine body as Fig. 5 d, the cancer suppressing action of dihydroarteannuin is better than arteannuin.Arteannuin and dihydroarteannuin all can increase the cancer suppressing action of gemcitabine, and associating gemcitabine therapeutic effect is better than single medicine, and especially dihydroarteannuin shows significant chemotherapy sensitization.In ovarian cancer cell A2780 and the experiment of OVCAR3 transplanted tumor in nude mice (Fig. 5 e), DHA (dosage be 10 with 25mg/kg) causes A2780 heteroplastic transplantation tumor 24% and 41% growth inhibited (comparing with the matched group of giving normal saline) (P＜0.05) respectively, and OVCAR-3 model 14% and 37% tumor growth suppress (P＜0.05); Be suppressed 56% (A2780) and 46% (OVCAR-3) only for the treatment group tumor growth of single CBP.Drug combination (25mg/kg DHA) group then all produces 70% tumor growth and suppresses (P＜0.05) in A2780 and OVCAR-3 animal tumor. Above cell and the experiment of animal level show, arteannuin and derivant thereof have the sensitization of good independent chemotherapy and associating one linearize treatment medicine, in conjunction with the artemisine derivative compound directly targeting PDGFRa bring into play this result of its anti-tumor activity, the prompting artemisinin-based drug can be used for clinical associating multiclass chemotherapeutics and treat tumour patient, and especially pathological diagnosis is the tumour patient of the PDGFRa positive Embodiment 4 PDGFR α is with pathological grading and shift relevant Present embodiment according to the clinical pathology diagnostic message strictness chosen 45 routine ovarian cancer patients' pathology sample, comprising 21 routine low level ovarian cancers (the clinical case rank is I-II, and moderate or highly differentiation do not have tangible lymph node, transfers such as abdominal cavity and nethike embrane); 24 routine high-level ovarian cancer patients (the clinical case rank is III-IV, and low differentiation has large-area lymphatic metastasis, abdominal cavity, nethike embrane, uterus, adnexa and other organ metastasis). The result shows (see figure 6), compares with the low level ovarian cancer, and in the high-level ovarian cancer case, the expression of tumor cell and Interstitial cell PDGFR α on every side significantly increases.Cell experiment shows, utilizes the gene perturbation technique of slow virus mediation that silence is carried out in the expression of PDGFR α among ovarian cancer cell A2780 and the OVCAR3, finds that the growth of tumor cell and migration invasive ability significantly reduce. Embodiment 5 Preparation of drug combination Compositions 1 Compositions 2 Discuss Existing discovery shows, PDGFR gene mutation or excessive activation can promote the transformation ability and then cause the generation of malignant tumor, as gastrointestinal stromal tumors (GISTs), and pulmonary carcinoma, carcinoma of prostate and breast carcinoma.Somatomedin such as PDGFR is combined the degraded that improves the iuntercellular adhesion molecule with its part, promote invasion by tumor cells and transfer, can also activate the VEGF in the microenvironment simultaneously, FGF and then promote angiogenesis have accelerated the malignant progression process of tumor.Zoopery and Clinical pathological study find that PDGFR α overexpression and activation have participated in generation and the evolution of ovarian cancer, with patient's pathology rank and poor prognosis significant correlation. Content of the present invention shows, compare with ovarian cancer patient's pathological tissue that low level shifts, the expression of PDGFR α significantly increases in extensive its cancerous cell of ovarian cancer patient that shifts of high-level generation and the mesenchyma stroma of tumors cell, this just shows that PDGFR α can be used as a target spot of ovarian cancer treatment. Find artemisinin derivatives among the present invention, especially arteannuin and dihydroarteannuin can directly be attached in the PDGFRa molecule, have both suppressed the receptor tyrosine kinase activity of PDGFRa, suppress the protein stability of PDGFRa again, promote its ubiquitin degraded. Molecule and zoopery show that the medicine of artemisine can be by suppressing multiple growth of tumor, migration and the invasive ability that PDGFRa and then inhibition comprise ovarian cancer and hepatocarcinoma.This result shows that artemisinin-based drug can be used as a kind of special inhibitor of PDGFR, is used for the tumour patient of PDGFRa sudden change clinically or excessive activation, comprises ovarian cancer, hepatocarcinoma, gastrointestinal stromal tumors (GISTs), acute leukemia etc.Artemisinin derivatives also has good anticancer sensitizing activity, for example artemisinin-based drug can significantly improve a line chemotherapeutic carboplatin, gemcitabines etc. are for the therapeutic effect of ovarian cancer and hepatocarcinoma, especially dihydroarteannuin has stronger chemotherapy sensitization to carboplatin and gemcitabine, its characteristics of high efficiency and low toxicity makes it to be expected to as chemotherapeutic sensitizer, is applied to the clinical combined chemotherapy of tumor. All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally. Claims (10) Hide Dependent 1. arteannuin and derivant thereof are for the preparation of suppressing to be selected from down the medicine of group disease or the purposes of medicine sensitizer: PDGFR α positive tumor, vascular conditions or fibrous lesions. 2. the purposes of an arteannuin and derivant thereof is characterized in that, for the preparation of inhibitor or the antagonist of platelet derived growth factor receptor A (PDGFR α). 3. purposes as claimed in claim 2 is characterized in that, described inhibitor or antagonist also are used for: (i) activation of inhibition PDGFR α tyrosine kinase; And/or The protein stability that (ii) suppresses PDGFR α; And/or (iii) promote the degraded of the proteasome mediation that PDGFR α ubiquitin relies on; And/or (vi) suppress the activation of PDGFR α positive tumor cell signal path PI3K/AKT and MAPK/EPK. 4. as the arbitrary described purposes of claim 1-2, it is characterized in that described artemisinin derivative is selected from down group: Artemether, dihydroartemisinine, artesunate, two hydrogen Artemether, arteether etc. 5. an external non-therapeutic ground suppresses the active method of platelet derived growth factor receptor A (PDGFR α), it is characterized in that, comprise step: with arteannuin and/or artemisinin derivative and cells contacting, thus platelet derived growth factor receptor A (PDGFR α) in inhibition or the antagonism cell. 6. the purposes of preparation combination, described preparation combination comprises: (I) contain the preparation of arteannuin and/or artemisinin derivative; With (II) contain the preparation of gemcitabine or carboplatin, It is characterized in that, the combination of described preparation for the preparation of: (a) pharmaceutical composition or the medicine box of inhibition platelet derived growth factor receptor A; Or (b) prevent and/or treat pharmaceutical composition or the medicine box of tumor. 7. purposes as claimed in claim 6 is characterized in that, described tumor is PDGFR α positive tumor. 8. method that prevents and/or treats the PDGFR alpha associated disorders, wherein said disease is relevant with expression excessively or the hyperactivity of PDGFR α, it is characterized in that, comprise step: use arteannuin and/or artemisinin derivative for the object of needs, or use the pharmaceutical composition that contains arteannuin and/or artemisinin derivative. 9. method, described method is used in the body or growth, migration and the invasion and attack of the activation of vitro inhibition PDGFR α tyrosine kinase, the protein stability of inhibition PDGFR α, the degraded that promotes the proteasome mediation that PDGFR α ubiquitin relies on, the activation that suppresses signal path PI3K/AKT and MAPK/EPK and/or inhibition PDGFR α positive tumor cell, it is characterized in that described method comprises step: use arteannuin and/or artemisinin derivative for the object of needs. 10. a test kit that is used for the ovarian cancer pathological grading is characterized in that, described test kit comprises the reagent that detects in vitro tissue PDGFR alpha expression amount. 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receptor A and application thereof https://patents.google.com/patent/CN103251585A/en 青蒿素及其衍生物在抑制血小板衍生生长因子受体A中的作用及其应用 技术领域 本发明涉及生物医药领域，具体地，本发明涉及青蒿素及其衍生物在抑制血小板衍生生长因子受体A中的作用及其应用。 背景技术 青蒿又名黄花蒿(Artemisia anaua L)，属菊科，是一年生草本植物。青蒿素是我国药学工作者于20世纪70年代初从中药青蒿中提取的倍半萜内酯类抗疟药物。鉴于传统的奎宁类药物普遍遇到抗药性问题，青蒿素和它的衍生物目前已取代它们，成为世界上抗疟疾的主流药物。青蒿素的大环上有一个内过氧化物桥，在亚铁离子或亚铁血红素的催化下，会放出以碳为中心的自由基。细胞内的蛋白质和核酸被这些自由基烷化后死亡。疟原虫寄生在红血球内，含有大量来自血红蛋白的亚铁血红素，因而青蒿素很易被活化，进而将疟原虫杀死。青蒿素和它的衍生物，如二氢青蒿素，蒿甲醚，蒿乙醚及青蒿琥酯等已广泛应用于疟疾治疗，更多的青蒿素衍生物正在研制中。 青蒿素及其衍生物的抗疟活性已得到世界公认，具有起效快、药效高、毒副作用低等优点。除广泛应用于抗疟治疗外，青蒿素类药物还具有其它多种药理作用，如抗血吸虫作用、抗心律失常、平喘、抗内毒素、抗变态反应、抗红斑狼疮、免疫抑制等作用。 随着对青蒿素及其衍生物活性研究的不断深入，人们发现，该类化合物对多种肿瘤细胞的生长具有抑制作用和良好的治疗效果，然而，其具体作用靶点及相关信号通路及作用机制还不明确。因此本领域迫切需要了解青蒿素及其衍生物在一直肿瘤中的作用。 发明内容 本发明的目的是提供青蒿素及其衍生物在抑制血小板衍生生长因子受体A中的作用。 本发明的另一目的是提供青蒿素及其衍生物作为血小板衍生生长因子受体A抑制剂/拮抗剂的应用。 本发明的另一目的是提供体外治疗肿瘤的方法。 在本发明的第一方面，提供了一种青蒿素及其衍生物用于制备抑制选自下组疾病的药物或药物增敏剂的用途：PDGFRα阳性肿瘤、血管性疾病、或纤维性病变。 在另一优选例中，所述的PDGFRα阳性肿瘤细胞为PDGFRα阳性卵巢癌细胞或PDGFRα阳性肝癌细胞。 在本发明的第二部分，提供了一种青蒿素及其衍生物的用途，用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂。 在另一优选例中，所述的血小板衍生生长因子受体A(PDGFRα)来自于人。 在另一优选例中，所述的抑制剂或拮抗剂还用于： (i)抑制PDGFRα酪氨酸激酶的活化；和/或 (ii)抑制PDGFRα的蛋白稳定性；和/或 (iii)促进PDGFRα泛素依赖的蛋白酶体介导的降解；和/或 (vi)抑制PDGFRα阳性肿瘤细胞信号通路PI3K/AKT和MAPK/EPK的活化。 在另一优选例中，所述的青蒿素衍生物选自下组：蒿甲醚、二氢青蒿素、青蒿琥酯、双氢蒿甲醚、蒿乙醚等。 在另一优选例中，所述的青蒿素衍生物为二氢青蒿素。 在本发明的第三方面，提供了一种体外非治疗性地抑制血小板衍生生长因子受体A(PDGFRα)活性的方法，包括步骤：将青蒿素和/或青蒿素衍生物与细胞接触，从而抑制或拮抗细胞中血小板衍生生长因子受体A(PDGFRα)。 在另一优选例中，所述的接触是在青蒿素和/或青蒿素衍生物存在下，培养所述细胞。 在另一优选例中，所述的细胞包括肿瘤细胞，较佳地包括PDGFRα阳性肿瘤细胞，更佳地包括PDGFRα阳性卵巢癌细胞或肝癌细胞，如卵巢癌细胞A2780、卵巢癌细胞OVCAR3、肝癌细胞Hep3B。 在另一优选例中，所述方法还用于至少以下一种应用： 体外非治疗性地抑制PDGFRα酪氨酸激酶的活化； 体外非治疗性地抑制PDGFRα的蛋白稳定性； 体外非治疗性地促进PDGFRα泛素依赖的蛋白酶体介导的降解； 体外非治疗性地抑制信号通路PI3K/AKT和MAPK/EPK的活化；和 体外非治疗性抑制PDGFRα阳性肿瘤细胞的生长。 在本发明的第四方面，提供了一种制剂组合的用途，所述制剂组合包括： (I)含青蒿素和/或青蒿素衍生物的制剂；和 (II)含吉西他宾或卡铂的制剂， 所述制剂组合用于制备： (a)抑制血小板衍生生长因子受体A的药物组合物或药盒；或 (b)预防和/或治疗肿瘤的药物组合物或药盒。 在另一优选例中，所述制剂包括：片剂、胶囊、栓剂、注射剂。 在另一优选例中，所述肿瘤为PDGFRα阳性肿瘤。 在本发明的第五方面，提供了一种预防和/或治疗PDGFRα相关疾病的方法，其中所述疾病与PDGFRα的过表达或活性过高有关，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物，或施用含青蒿素和/或青蒿素衍生物的药物组合物。 在另一优选例中，所述的PDGFRα相关疾病包括肿瘤(PDGFRα阳性肿瘤)，如卵巢癌或肝癌。 在另一优选例中，PDGFRα阳性肿瘤为PDGFRα阳性卵巢癌或PDGFRα阳性肝癌。 在另一优选例中，所述的施用包括：瘤内、静脉注射、口服片剂、腹腔注射等。 在另一优选例中，所述的对象为哺乳动物。 在本发明的第六方面，提供了一种方法，所述方法用于体内或体外抑制PDGFRα酪氨酸激酶的活化、抑制PDGFRα的蛋白稳定性、促进PDGFRα泛素依赖的蛋白酶体介导的降解、抑制信号通路PI3K/AKT和MAPK/EPK的活化、和/或抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物。 在本发明的第七方面，提供了一种用于卵巢癌病理分级的试剂盒，所述试剂盒包含检测离体组织PDGFRα表达量的试剂。 应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。 附图说明 下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。 图1显示PDGFRα可以作为青蒿素及其衍生物的直接作用靶标；其中，图1a为体外激酶实验，结果显示，青蒿素及其衍生物青蒿素(ART)、二氢青蒿素(DHA)、青蒿琥酯(ARM)、蒿甲醚(ARS)可以抑制PDGFRα胞内端(aa550-末端)酪氨酸激酶的活性；图1b为表面等离子共振(SPR)实验，结果显示，双氢青蒿素(DHA)可以与PDGFRα包外端(aa24-524)蛋白相互结合，索拉菲尼(Sorafenib)为阳性对照，已知其可以结合抑制PDGFRα的活性；图1c为生物素-亲和素亲和纯化实验，结果显示，标记生物素的DHA可以与A2780细胞内PDGFRα相互结合，从而被亲和纯化出来；图1d为计算机辅助同型模建及反向对接实验(ComputerAssisted Homology modeling and docking)，结果显示，双氢青蒿素(DHA)可以嵌合到PDGFRα包外端PDGF结合位点，同时也可嵌合到其包内端ATP结合位点，阻断其激酶激活；图1e-图1f显示双氢青蒿素能够有效抑制PDGFRα阳性细胞A2780和OVCAR3中PDGFRα的活性和蛋白表达，而对PDGFRβ的磷酸化和蛋白水平影响不大；图1g-图1i显示双氢青蒿素可以加速PDGFRα的蛋白降解，这一过程是泛素依赖的蛋白酶体介导的。 图2显示了青蒿素及其衍生物具有抑制PDGFRa阳性的卵巢癌细胞的生长和迁移能力；其中，图2a显示，给予药物刺激48h后，青蒿素及其衍生物对人正常卵巢上皮细胞IOSE144无明显的生长抑制能力，对于SK-OV3细胞毒性较小，但对于PDGFRa阳性的A2780和OVCAR3具有较强抑制能力，其中青蒿素和二氢青蒿素的作用最为显著；图2b为细胞迁移实验，结果显示，低浓度短时间处理(12h)卵巢癌细胞，青蒿素及其衍生物能够有效地抑制PDGFRa阳性细胞A2780的迁移能力，而对于PDGFRa阴性的细胞SK-OV3无明显抑制能力；图2c显示了不同浓度的双氢青蒿素对5种细胞系(IOSE144、A2780、OVCAR3、OVCAR5、SK-OV3)的细胞抑制作用；图2d显示了不同浓度的双氢青蒿素对3种细胞系(A2780、OVCAR3、SK-OV3)的细胞迁移的影响；图2e显示了不同浓度的双氢青蒿素对2种细胞系(A2780、OVCAR3)的细胞侵袭的影响。 图3显示青蒿素及其衍生物具有抑制PDGFRa阳性的肝癌细胞的生长和迁移能力；其中，图3a显示，在人正常的肝细胞系(7702和LO2)和肝癌细胞系(Hep3B、HepG2、7721、7404、LM3、97L、97M、Huh-7)中，只有Hep3B高表达PDGFRa；图3b显示，低浓度双氢青蒿素(3μM)短时(12h)处理Hep3B细胞，能够显著抑制其迁移和侵袭能力；图3c为细胞生长实验，结果显示，PDGFRa阳性的Hep3B细胞对于双氢青蒿素更敏感，而其他细胞对于双氢青蒿素敏感程度较低。 图4显示双氢青蒿素能抑制小鼠原位卵巢癌模型的生长和转移；其中，图4a显示双氢青蒿素给予治疗小鼠，能有有效地抑制卵巢癌的恶性进展；图4b显示，与对照组相比，双氢青蒿素治疗组小鼠的肺癌转移的情况大大降低，且随着药量的增加，治疗情况越好；图4c显示不同浓度的双青青蒿素给予治疗小鼠，肿瘤腹腔播散的情况(肺脏、肝脏、肠)得到明显的抑制；图4d为免疫组化实验，结果显示，双氢青蒿素处理的细胞其形态转换过程(EMT)得到抑制；图4e为免疫印迹实验，结果显示，双氢青蒿素能够有效抑制PDGFRa的表达和磷酸化，下游信号通路PI3K/AKT和MAPK-ERK得以抑制。 图5显示了青蒿素及其衍生物与一线化疗药的增敏作用；其中，图1a为HepG2和Hep3B细胞用青蒿素或双氢青蒿素单独处理或联合吉西他宾处理实验，结果显示，吉西他宾单独作用于HepG2和Hep3B细胞，具有一定的凋亡促进作用，青蒿素联合吉西他宾对HepG2和Hep3B细胞都具有显著凋亡促进作用作用，两药联合表现出协同作用；图5b显示，DHA和CBP联合用药24小时后对OVCAR-3细胞凋亡有增效促进作用，用1μM的DHA联合500μM CBP(1155％)作用后的细胞凋亡率显著高于单独用1μM DHA(266％)或500μM CBP(528％)的细胞凋亡率，而且超过了两种化合物的凋亡累加效应，是一种增效作用，A2780细胞对联合治疗显示了累加效应；图5c显示，当与DHA联用时，CBP显著抑制了卵巢癌细胞的活力，当细胞暴露于1μM CBP和1μM DHA时，A2780细胞的存活率就降低了69％，而OVCAR-3细胞存活则减少了72％，相比之下，IOSE144细胞则显得对治疗较不敏感，两种药物均以1μM合用时存活率只降低了约28％；图5d为肝癌细胞Hep3B和HepG2裸鼠移植瘤实验，结果显示，青蒿素和双氢青蒿素单药体内均具有抗肿瘤作用，双氢青蒿素的抑癌作用强于青蒿素，青蒿素及双氢青蒿素均能增加吉西他宾的抑癌作用，联合吉西他宾治疗效果优于单药，尤其是双氢青蒿素表现出显著的化疗致敏作用；图5e为在卵巢癌细胞A2780和OVCAR3裸鼠移植瘤实验中，结果显示，DHA(剂量为10和25mg/kg)分别导致A2780异体移植肿瘤24％和41％的生长抑制(与给生理盐水的对照组相比)(P＜0.05)，OVCAR-3模型14％和37％肿瘤生长抑制(P＜0.05)；只给单一CBP的治疗组肿瘤生长被抑制了56％(A2780)和46％(OVCAR-3)，联合用药(25mg/kgDHA)组则在A2780和OVCAR-3动物肿瘤中均产生70％肿瘤生长抑制(P＜0.05)。 图6显示PDGFRα的表达与卵巢癌病理级别和转移状态的关系；其中，图6a显示，与低级别卵巢癌相比，高级别卵巢癌病例中，肿瘤细胞及周围的间质细胞PDGFRα的表达量显著增加；图6b为PDGFRα染色结果，结果显示，在高级别卵巢癌病例中，PDGFRα的表达显著增加。 具体实施方式 本发明人经过广泛而深入的研究，发现青蒿素及其衍生物的用途，用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂，此外，青蒿素及其衍生物还可以抑制PDGFRα酪氨酸激酶的活化、抑制PDGFRα的蛋白稳定性、促进PDGFRα泛素依赖的蛋白酶体介导的降解、抑制信号通路PI3K/AKT和MAPK/EPK的活化、抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭、还可以用于PDGFRα阳性肿瘤治疗的增敏剂。在此基础上完成了本发明。 术语 卵巢癌 卵巢癌是全世界的妇女健康的巨大威胁。在美国，卵巢癌已成为第四位致死性的女性癌症。虽然被诊断于病发早期的卵巢癌患者的5年存活率可以达到80％-90％，但是被诊断为晚期的卵巢癌患者存活率却只有不到25％，此外，大多数卵巢癌患者在诊断出是卵巢癌是已至晚期。目前卵巢癌患者的死亡率在过去的数十年没有发生太大的改变，因此在肿瘤治疗和预后中，寻找有效的治疗方案，增强肿瘤细胞对化疗药物的敏感性，和克服肿瘤的耐药性是研究人员和临床实践人员长期以来的目标。 肝癌 肝癌是最常见的恶性肿瘤之一，我国是肝癌的高发区，其发病率已上升到恶性肿瘤的第二位。由于肝癌早期症状不明显，因此确诊的大多数病人已处于中、晚期。原发性肝癌的首选治疗是手术切除，随着治疗技术的提高，目前术后5年生存率已提高至50％-60％，但由于手术切除只适用直径不超过5cm、癌灶数目不超过3个的小肝癌。此外，是否适合直接手术切除还取决于癌灶部位的血管富集程度及病人个体差异，这些限制导致90％左右的肝癌不适合手术治疗。即使是可切除肝癌，术后实施以介入化疗为主的综合性治疗也是必要的。然而目前，鲜有药物能够有效抑制肝癌的生长和进一步恶化，肝癌对传统化疗药物的反应率低，毒副作用非常显著，因此化疗的缓慢发展是制约抗肿瘤药物临床应用的主要原因。因此对于肿瘤化疗来说，迫切需要寻求更好的化疗增效剂和更好的治疗方案，从而提高肿瘤对化疗的敏感度，降低其对化疗的耐药性。 血小板衍生生长因子及其受体 血小板衍生生长因子(Platelet derived growth factor，PDGF)是一类能够合成并分泌到细胞外基质中的生长因子，具有促进纤维细胞、平滑肌细胞等多重组织的细胞分裂、增殖、迁移、增加细胞的黏附能力，在人胚胎发育和正常生理活动中发挥着重要作用。PDGF以自分泌和旁分泌形式激活血小板衍生生长因子受体(PDGFR)而发挥作用。 PDGFR包含两种结构类似的酪氨酸激酶受体PDGFRα和PDGFRβ，其中PDGFRα的异常激活与多重疾病密切相关，具体地，PDGFR相关疾病可以分为三类： 1.肿瘤 由于PDGFR(特别是PDGFRα)存在于多种肿瘤中，它可能作为一个新的治疗靶点。已经发现，多种肿瘤的发生与PDGFR(特别是PDGFRα)的过度激活相关，如卵巢癌、神经胶质瘤、前列腺癌；此外，在一些肿瘤中存在PDGFR的突变激活，如隆突性皮肤纤维肉瘤、胃肠道间质瘤、Bcr-Abl阴性的慢性髓样白血病、嗜酸细胞增多综合征等等。 2.血管性疾病 研究发现，PDGF及PDGFR等在多种血管性疾病中存在过量表达和过度激活现象，如动脉粥样硬化和再狭窄、肺动脉高压、视网膜血管病变等，抑制PDGFR信号通路的激活可以有效缓解上述疾病的症状，增加其治疗效果。 3.纤维性病变 PDGF-PDGFR信号通路在多种纤维性病变过程中发挥重要的作用，如肺纤维化、肝纤维化和肝硬化、皮肤纤维增生(硬皮病)、肾纤维化、心肌纤维化等等，其中PDGF-PDGFR信号通路介导的间质细胞的增殖是该慢性炎症的共同特征。 上述三类疾病中，PDGF-PDGFR信号通路发挥重要的作用，其中PDGFRβ主要介导了血管系统病变，而PDGFRα主要介导了间质细胞和成纤维细胞驱动的病变。(Johanna Andrae，et al.Role of platelet-derived growth factors inphysiology and medicine，Genes Dev.2008；22：1276-1312；张秀华等，血小板源生长因子受体与肿瘤.生命科学，Vol.18，No.3 Jun.，2006) 青蒿素及其衍生物 青篙素是从中药青篙中提取的有过氧基团的倍半萜内酯药物，由于青蒿素是复杂的大分子，化学全人工很难合成，因此目前主要是生物合成和人工提取。 青蒿素等倍半萜类的生物合成在细胞质中进行，途径属于植物类异戊二烯代谢途径，可分为三大步：由乙酸形成FPP，合成倍半萜，再内酯化形成青蒿素，具体为：FPP→4，11-二烯倍半萜→青蒿酸→二氢青蒿酸→二氧青蒿酸过氧化物→青蒿素。 青蒿素和本领域几种常见的青蒿素衍生物的化学式见下： 青蒿素及其衍生物的应用 本发明提供了青蒿素及其衍生物用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂的用途。 在另一优选例中，青蒿素及其衍生物还用于： (i)抑制PDGFRα酪氨酸激酶的活化；和/或 (ii)抑制PDGFRα的蛋白稳定性；和/或 (iii)促进PDGFRα泛素依赖的蛋白酶体介导的降解；和/或 (vi)抑制PDGFRα阳性肿瘤细胞信号通路PI3K/AKT和MAPK/EPK的活化；和/或 (v)抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭；和/或 (vi)制备抑制PDGFRα阳性肿瘤的药物或增敏剂；和/或 (vii)制备治疗血管性疾病和纤维性病变的药物或增敏剂。 在本发明的一个优选例中，提供了一种体外非治疗性地抑制血小板衍生生长因子受体A(PDGFRα)活性的方法，包括步骤：将青蒿素和/或青蒿素衍生物与细胞接触，从而抑制或拮抗细胞中血小板衍生生长因子受体A(PDGFRα)。 所述的接触是在青蒿素和/或青蒿素衍生物存在下，培养所述细胞。细胞包括肿瘤细胞，较佳地包括PDGFRα阳性肿瘤细胞，更佳地包括PDGFRα阳性卵巢癌细胞或肝癌细胞，如卵巢癌细胞A2780、卵巢癌细胞OVCAR3、肝癌细胞Hep3B。在另一优选例中，所述方法还用于至少以下一种应用：体外非治疗性地抑制PDGFRα酪氨酸激酶的活化；体外非治疗性地抑制PDGFRα的蛋白稳定性；体外非治疗性地促进PDGFRα泛素依赖的蛋白酶体介导的降解；体外非治疗性地抑制信号通路PI3K/AKT和MAPK/EPK的活化；和体外非治疗性抑制PDGFRα阳性肿瘤细胞的生长。 本发明还提供了一种预防和/或治疗PDGFRα相关疾病的方法，其中所述疾病与PDGFRα的过表达或活性过高有关，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物，或施用含青蒿素和/或青蒿素衍生物的药物组合物。PDGFRα相关疾病包括肿瘤(PDGFRα阳性肿瘤)，如卵巢癌或肝癌。在另一优选例中，所述的施用包括：瘤内、静脉注射、口服片剂、腹腔注射等。所述的对象为哺乳动物(如人)。 药剂组合物及其应用 本发明还提供了药剂组合物，在一个优选例中，所述制剂组合包括： (I)含青蒿素和/或青蒿素衍生物的制剂；和(II)含吉西他宾或卡铂的制剂， 所述制剂组合用于制备：(a)抑制血小板衍生生长因子受体A的药物组合物或药盒；或(b)预防和/或治疗肿瘤的药物组合物或药盒。 所述制剂包括片剂、胶囊、栓剂、注射剂；所述药物制剂用于预防和治疗PDGFRα阳性肿瘤。 本发明的药剂组合物可以是固体或液体。固体制剂包括散剂、片剂、丸剂、胶囊、扁囊剂、栓剂和可分散颗粒剂，固体载体可以是一种或多种物质，它们可以作为稀释剂、矫味剂、黏合剂、防腐剂、片剂崩解剂或包囊材料。在散剂中，载体是微细分的固体，本发明的化合物与微细分的活性组份存在于混合物中。在片剂中，此化合物与所需的粘合载体以适当比例混合，并压制成所需的形状和大小。散剂和片剂中优先含有5至70％活性化合物，适宜的载体是碳酸镁、硬脂酸镁、滑石、糖、乳糖、果胶、糊精、淀粉、明胶、羧甲基纤维素、羧甲基纤维素钠、低熔点腊和可可脂等。同样，扁囊剂或锭剂、片剂、散剂、胶囊、丸剂、扁囊剂和锭剂可以是适合于口服给药的固体剂型。为了制备栓剂，可以将低熔点腊乳脂肪酸甘油脂或可可脂的混合物熔化，并通过搅拌将此活性化合物组份均匀地分散在其中，然后将熔化的均匀混合物倒入大小合适的模中让其冷却，并由此固化。溶液形式的制剂包括溶液、悬浮剂和乳剂，例如水或含水丙二醇溶液。对于非肠道注射液体制剂，可以在含水聚乙二醇溶液中制备。适于口服的含水溶液可以通过将活性组份溶解于水中，并按照需要加入适宜的着色剂、矫味剂、乳化剂和增稠剂制备。适于口服的含水混悬剂可以通过将微细分的活性组份分散于含水粘性物质中制备，如天然或合成胶、树脂、甲基纤维素、羟甲基纤维素钠及其他熟知的混悬剂。这些液体形式包括溶液、混悬剂和乳剂，除活性组份外，这些制剂可含有着色剂、矫味剂、稳定剂、缓冲剂、合成或天然甜味剂、分散剂、增稠剂和助溶剂等。此药物制剂优先是单位剂型，在此剂型中，此制剂被再分为含适量活性组份的单位剂量，此单位剂量可以是包装的制剂，该包装含有一定量的制剂，如包装的片剂、胶囊和在小瓶或胶囊中的散剂。此单位剂型还可以是胶囊、片剂、扁囊剂或锭剂本身，或其可以存在包装形式中的适当数量的任何这些散剂。 单位剂量制剂中活性组份的量可根据具体的应用和活性组份的效力而变化，对于非人哺乳动物(如小鼠)治疗，可以将0.01mg至约0.5g，较佳地0.1至约3mg的胶囊每天给药三次，必要时该组合物还可以含其他相容的治疗剂。对于人的治疗，可以根据患者的需要、被治疗病症的严重性以及使用的化合物而变化，较佳地，开始以小于该化合物最佳剂量的较小剂量治疗，此后，小量增加此剂量达到最佳效果，方便起见，如果需要可将总日剂量再细分为一天内分次给药。在本发明的一个优选例中，给予人的剂量大约为0.1/kg-0.5g/kg之间。 本发明的主要优点： (1)青蒿素及其衍生物可以用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂； (2)青蒿素及其衍生物可以抑制PDGFRα酪氨酸激酶的活化、PDGFRα的蛋白稳定性、抑制信号通路PI3K/AKT和MAPK/EPK的活化、抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭； (3)青蒿素及其衍生物可以促进PDGFRα泛素依赖的蛋白酶体介导的降解； (4)青蒿素及其衍生物可以用于PDGFRα阳性肿瘤治疗的增敏剂。 下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：ColdSpring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。 实验方法和材料 1.表面等离子共振 表面等离子共振(SPR)是一种光学现象，可被用来实时跟踪在天然状态下生物分子间的相互作用。实验中，将重组人PDGFRα胞外端(Met1-Glu524)蛋白，键合在生物传感器表面，将不同浓度的双氢青蒿素溶液和一直与PDGFRα有相互作用的化合物索拉菲尼及SU11248溶液注入并流经生物传感器表面。生物分子间的结合引起生物传感器表面质量的增加，导致折射指数按同样的比例增强，生物分子间反应的变化即被观察到，这种反应用反应单位(RU)来衡量。 2.细胞培养 人卵巢永生化的非致瘤性上皮细胞IOSE-144和卵巢癌细胞(A2780，OVCAR-3，SK-OV3，OVCAR-5)购自美国ATCC(American Type CultureCollection，Manassas，VA)。所有细胞依据ATCC的培养要求(RPMI-1640或DMEM培养液加入10％胎牛血清(GIBCO)、100IU/ml青霉素、100IU/ml链霉素)于5％CO2的37℃培养箱中培养。 3.细胞生长抑制实验 细胞生长及增殖抑制试验通过CCK-8实验测定。细胞以3×103个细胞每孔的密度铺种于96孔板中。12～18小时后，以一定浓度梯度(0、1、5、10、25μM)的青蒿素类化合物处理48小时之后，在96孔板每孔中加10μL CCK-8溶液进行药物处理后的细胞生长活性评价，实验最后通过酶标仪(SpectraMax190microplate reader；Molecular Devices，USA)测量450nm处的光吸收(OD值)(每实验浓度至少设置三个复孔，每次实验至少重复三次)。 4.细胞迁移、侵袭实验 卵巢癌细胞的迁移和侵袭能力通过transwell实验测定，低浓度青蒿素衍生物短时间(12h)处理卵巢癌细胞，然后将相同数目的细胞(5×104个)重悬在无血清的培养基中，种植到transwell(测定细胞的侵袭能力时，transwell上层铺上基质胶)上层培养室中。以10％胎牛血清培养基作为化学趋化剂。大约8-12h后，未迁移的细胞用棉签擦去，将迁移的细胞进行固定染色，并进行统计分析。 5.SDS-聚丙烯酰胺凝胶电泳和蛋白免疫印迹分析 以不同浓度的DHA处理培养细胞24小时，收集细胞并用细胞裂解液(RIPAbuffer #9806，Cell Signaling)裂解。之后以13,000转于4℃离心15min，取上清进行后续的分析检测。蛋白浓度通过Bio-Rad公司的试剂盒(protein assay kit；Hercules，CA)定量蛋白总量。之后每孔上等量蛋白进行SDS-PAGE胶电泳分离。电泳完毕，将电泳蛋白条带转移至甲醇活化过的PVDF膜(Millipore，Bedford，MA)上。转膜完毕后，膜以5％的脱脂牛奶(PBS溶解)封闭，然后后加一抗和二抗孵育(以5％的BSA配置)，之后通过蛋白条带表达差异变化以蛋白显影试剂盒(ECL plus system，Amersham Pharmacia Biotech)显影分析。 6.小鼠原位卵巢癌异体移植瘤模型体内治疗实验 四到六周龄的雌性BALB/c(nu/nu)购自上海斯莱克实验动物有限公司(Shanghai Experimental Animal Center)，并按规定饲养于SPF级动物房。所有动物实验操作均得到中科院营养所动物伦理委员会的批准。。大体步骤如下：将荧光素酶标记的A2780细胞，重悬于无血清的RPMI 1640培养基中。然后腹腔注射等量细胞(～3×106 cells/0.2ml)于小鼠的腹腔内部。利用IVIS Lumina生物荧光系统实时监测肿瘤的生长情况，并记录小鼠的体重。注射5天后，根据肿瘤的荧光强度时将小鼠平均分成治疗组和对照组(每组8只老鼠)。治疗组药物DHA溶解在蓖麻油、乙醇和生理盐水的混合物(CremophorEL∶Ethanol∶Saline＝5∶5∶90，v/v/v)中，所有药物均通过腹腔给药。DHA的给药剂量为10或25mg/kg体重，给药频率为每天给药，每周停歇两天。对照组注射生理盐水。在治疗过程中利用活体成像系统实时监测肿瘤的生长状况。治疗结束后，小心取出移腹部肿瘤(除掉脂肪和结缔组织)，以PIPA溶液(100mg组织加入1ml RIPA)匀浆。得到的肿瘤组织匀浆液按上述的蛋白免疫印迹分析实验要求进行处理，之后进行蛋白免疫印迹检测；或将肿瘤组织进行固定和免疫染色。 实施例1 青蒿素及其衍生物直接结合PDGFRα，抑制其磷酸化活性，促进其降解 利用PDGFRα体外激酶实验、表面等离子共振、计算机辅助同型模建及反向对接实验等分子药理学以及生物物理学等先进的生物学技术和手段，证实青蒿素类衍生物可以直接结合PDGFRα，抑制PDGFRα的受体酪氨酸激酶活性，同时研究发现双氢青蒿素可以抑制PDGFRα阳性细胞中PDGFRα的蛋白稳定性，促进其泛素依赖的降解过程。 图1表明，青蒿素及其衍生物能够直接结合PDGFRα，PDGFRα作为青蒿素及其衍生物作用的直接靶标，且能够抑制其磷酸化活性，促进其降解。体外激酶实验显示青蒿素及其衍生物：青蒿素(ART)、二氢青蒿素(DHA)、青蒿琥酯(ARM)、蒿甲醚(ARS)可以抑制PDGFRα的胞内端(aa550-末端)酪氨酸激酶的活性(图1a)。表面等离子共振(SPR)实验显示，双氢青蒿素(DHA)可以与PDGFRα包外端(aa24-524)蛋白相互结合，索拉菲尼(Sorafenib)做为阳性对照，已知可以结合抑制PDGFRα的活性(图1b)。生物素-亲和素亲和纯化实验显示标记生物素的DHA可以与A2780细胞内PDGFRα相互结合，从而被亲和纯化出来(图1c)。计算机辅助同型模建及反向对接实验(Computer Assisted Homology modeling anddocking)实验显示双氢青蒿素(DHA)可以嵌合到PDGFRα包外端PDGF结合位点，同时也可嵌合到其包内端ATP结合位点，阻断其激酶激活(图1d)。图1e-图1f显示了双氢青蒿素能够有效抑制PDGFRα阳性细胞A2780和OVCAR3中PDGFRα的活性和蛋白表达，而对PDGFRβ的磷酸化和蛋白水平影响不大。图1g-图1i实验显示双氢青蒿素可以加速PDGFRα的蛋白降解，这一过程是泛素依赖的蛋白酶体介导的。 实施例2 青蒿素及其衍生物抑制PDGFRα阳性的肿瘤细胞的生长和转移 利用裸鼠原位卵巢癌异体移植瘤模型，研究双氢青蒿素在动物体内的抗卵巢癌活性。将荧光素酶标记的人卵巢癌细胞A2780注射入Balb-c裸鼠的腹腔，建立裸鼠卵巢癌原位模型，然后给予双氢青蒿素治疗：对照组(生理盐水)，低剂量组(DHA 10mg/kg)。高剂量组(DHA 25mg/kg)每周治疗五次，停歇两天，共给予6周治疗时间。期间利用活体成像系统观察小鼠卵巢癌的生长和转移情况。 通过细胞水平和动物水平的一系列研究，显示青蒿素类衍生物能够有效地抑制PDGFRα阳性的肿瘤细胞的生长、迁移和侵袭(卵巢癌和肝癌)，而对于不表达PDGFRα的肿瘤细胞生长和迁移能力的抑制能力较小。具体结果如下： 图2表明，青蒿素及其衍生物抑制PDGFRa阳性的卵巢癌细胞的生长和迁移能力。具体地，如图1e和图2a所示，PDGFRa在卵巢正常细胞上皮细胞IOSE144，卵巢癌细胞SK-OV3，OVCAR5中无表达，在卵巢癌细胞A2780和OVCAR3中高表达；给予药物刺激48h后，不同浓度的青蒿素及其衍生物人正常卵巢上皮细胞IOSE144无明显的生长抑制能力，对于SK-OV3细胞毒性较小，但对于PDGFRa阳性的A2780和OVCAR3具有较强抑制能力，其中青蒿素和二氢青蒿素的作用最为显著。细胞迁移实验显示低浓度短时间处理(12h)卵巢癌细胞，青蒿素及其衍生物能够有效地抑制PDGFRa阳性细胞A2780的迁移能力，而对于PDGFRa阴性的细胞SK-OV3无明显抑制能力(图2b)。青蒿素衍生物中双氢青蒿素对于PDGFRa阳性的细胞的生长和迁移侵袭能力抑制最为明显(图2c-图2e)。 肝癌细胞中也得到了类似的结果，图3表明，青蒿素及其衍生物能够抑制PDGFRa阳性的肝癌细胞的生长和迁移能力。具体地，在众多人正常肝细胞(7702和LO2)和肝癌细胞系(Hep3B，HepG2，7721，7404，LM3，97L，97M，Huh-7)中，Hep3B高表达PDGFRa(图3a)；低浓度双氢青蒿素(3μM)短时(12h)处理Hep3B细胞能够显著抑制其迁移和侵袭能力(图3b)；细胞生长实验同时显示PDGFRa阳性的Hep3B细胞对于双氢青蒿素更为敏感，而其他PDGFRa地表达和无表达的肝癌细胞对于双氢青蒿素敏感程度较低(图3c)。 动物实验评估结果(图4)表明，双氢青蒿素对于PDGFRa阳性细胞A2780的生长和转移的具有抑制能力。具体地，双青青蒿素给予治疗后能有有效地抑制卵巢癌的恶性进展(图4a)；与对照组别相比，给予双氢青蒿素治疗组小鼠的为转移的情况大大降低，肿瘤腹腔播散的情况也得到明显的抑制(图4b、图4c)；免疫组化和免疫印迹实验同时显示，双氢青蒿素能够有效地抑制PDGFRa的表达和磷酸化，下游信号通路PI3K/AKT和MAPK-ERK得以抑制，上皮间质形态转换过程(EMT)得到抑制(图4d、图4e)。 实施例3 青蒿素及其衍生物与一线化疗药的增敏作用 青蒿素及双氢青蒿素体外单独应用均能够显著抑制卵巢癌和肝癌细胞的生长、增殖和迁移侵袭能力。本实施例对其是否具有化疗增敏作用进行了检测，青蒿素及双氢青蒿素分别联合吉西他宾作用于人肝癌细胞HepG2和Hep3B，以及联合卡铂作用于卵巢癌A2780和OVCAR3观察联合用药对细胞生长的抑制作用。结果(图5)如下所示： HepG2，Hep3B细胞用10μmol/L青蒿素或双氢青蒿素单独处理或联合10μg/L吉西他宾处理48小时，按上述方法进行细胞凋亡定量检测。具体地，吉西他宾单药作用于HepG2，Hep3B细胞显示出一定的凋亡促进作用(图5a)，青蒿素联合吉西他宾对HepG2，Hep3B细胞具有显著凋亡促进作用作用，两药联合表现出协同作用(图5a)。DHA和CBP联合用药24小时后对OVCAR-3细胞凋亡有增效促进作用(图5b)；用1μM DHA联合500μM CBP(1155％)作用后的细胞凋亡率显著高于单独用1μM DHA(266％)或500μM CBP(528％)的细胞凋亡率，而且超过了两种化合物的凋亡累加效应，显示出的是一种潜在的增效作用。然而，A2780细胞对联合治疗则显示的是累加效应而不是增效效应。这可能是由于它们对DHA化合物的敏感度较低的缘故(图5b，P＜0.05)。更长时间的作用可能产生更好的效果。在细胞增殖活力抑制方面，当与DHA联用时，CBP非常显著地抑制了卵巢癌细胞的活力。事实上，当细胞暴露于1μM CBP和1μM的DHA时，A2780细胞的存活率降低了69％，而OVCAR-3细胞存活则减少了72％。相比之下，IOSE144细胞则显得对治疗较不敏感，两种药物均以1μM合用时存活率只降低了约28％(图5c)。 细胞实验证明青蒿素以及双氢青蒿素在体外都显示出对于吉西他宾，卡铂处理癌细胞的杀伤和诱导凋亡的增敏作用。此外，裸鼠移植瘤实验进一步证明它们对于体内肿瘤的化疗也一样有效。具体地，如图5d显示，在肝癌细胞Hep3B和HepG2裸鼠移植瘤实验中，青蒿素和双氢青蒿素单药体内均具有抗肿瘤作用，双氢青蒿素的抑癌作用强于青蒿素。青蒿素及双氢青蒿素均能增加吉西他宾的抑癌作用，联合吉西他宾治疗效果优于单药，尤其是双氢青蒿素表现出显著的化疗致敏作用。在卵巢癌细胞A2780和OVCAR3裸鼠移植瘤实验中(图5e)，DHA(剂量为10和25mg/kg)分别导致A2780异体移植肿瘤24％和41％的生长抑制(与给生理盐水的对照组相比)(P＜0.05)，OVCAR-3模型14％和37％肿瘤生长抑制(P＜0.05)；只给单一CBP的治疗组肿瘤生长被抑制了56％(A2780)和46％(OVCAR-3)。联合用药(25mg/kg DHA)组则在A2780和OVCAR-3动物肿瘤中均产生70％肿瘤生长抑制(P＜0.05)。 以上细胞和动物水平实验显示，青蒿素及其衍生物具有良好的单独化疗和联合一线化疗药物的增敏作用，结合青蒿素类衍生化合物可以直接靶向PDGFRa发挥其抗肿瘤活性这一结果，提示青蒿素类药物可以用于临床中联合多类化疗药物来治疗肿瘤病人，尤其是病理诊断为PDGFRa阳性的肿瘤病人 实施例4 PDGFRα与病理分级和转移相关 本实施例根据临床病理诊断信息严格选取了45例卵巢癌病人的病理样本，其中包括21例低级别卵巢癌(临床病例级别为I-II，中度或高度分化，无明显的淋巴结，腹腔和网膜等转移)；24例高级别卵巢癌病人(临床病例级别为III-IV，低分化，具有大面积的淋巴结转移，腹腔，网膜，子宫，附件及其他器官转移)。 结果显示(见图6)，与低级别卵巢癌相比，高级别卵巢癌病例中，肿瘤细胞及周围的间质细胞PDGFRα的表达量显著增加。细胞实验显示，利用慢病毒介导的基因干扰技术将卵巢癌细胞A2780和OVCAR3中PDGFRα的表达进行沉默，发现肿瘤细胞的生长和迁移侵袭能力显著降低。 实施例5 药物组合物的制备 组合物1 组合物2 讨论 已有发现表明，PDGFR基因突变或过度激活可以促进细胞的转化能力进而导致恶性肿瘤的发生，如胃肠间质瘤，肺癌，前列腺癌和乳腺癌。PDGFR与其配体结合提高细胞间黏附分子的降解，促进肿瘤细胞的侵袭和转移，同时还能激活微环境中的VEGF，FGF等生长因子进而促进血管生成，加速了肿瘤的恶性进展过程。动物实验和临床病理研究发现，PDGFRα过度表达和激活参与了卵巢癌的发生和发展过程，与病人的病理级别和不良预后显著相关。 本发明内容表明，与低级别未发生转移的卵巢癌病人病理组织相比，高级别发生大规模转移的卵巢癌病人其癌细胞和肿瘤间质细胞中PDGFRα的表达量显著增加，这就表明，PDGFRα可以作为卵巢癌治疗的一个靶点。 本发明中发现青蒿素类衍生物，尤其是青蒿素和双氢青蒿素可以直接结合到PDGFRa分子中，既抑制PDGFRa的受体酪氨酸激酶活性，又抑制PDGFRa的蛋白稳定性，促进其泛素化降解。 分子和动物实验显示，青蒿素类的药物可以通过抑制PDGFRa进而抑制包括卵巢癌和肝癌在内的多重肿瘤的生长、迁移和侵袭转移能力。该结果表明，青蒿素类药物可以作为PDGFR的一种特殊的抑制剂，用于临床上PDGFRa突变或过度激活的肿瘤病人，包括卵巢癌、肝癌、胃肠间质瘤、急性白血病等等。青蒿素类衍生物也具有良好的抗癌增敏活性，例如青蒿素类药物可以显著提高一线化疗药卡铂，吉西他滨等对于卵巢癌和肝癌的治疗效果，尤其是双氢青蒿素对卡铂和吉西他宾具有更强的化疗致敏作用，其高效低毒的特点使之有望作为化疗增敏剂，应用于肿瘤的临床联合化疗。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 Claims (10) 1.青蒿素及其衍生物用于制备抑制选自下组疾病的药物或药物增敏剂的用途：PDGFRα阳性肿瘤、血管性疾病、或纤维性病变。 2.一种青蒿素及其衍生物的用途，其特征在于，用于制备血小板衍生生长因子受体A(PDGFRα)的抑制剂或拮抗剂。 3.如权利要求2所述的用途，其特征在于，所述的抑制剂或拮抗剂还用于： (i)抑制PDGFRα酪氨酸激酶的活化；和/或 (ii)抑制PDGFRα的蛋白稳定性；和/或 (iii)促进PDGFRα泛素依赖的蛋白酶体介导的降解；和/或 (vi)抑制PDGFRα阳性肿瘤细胞信号通路PI3K/AKT和MAPK/EPK的活化。 4.如权利要求1-2任一所述的用途，其特征在于，所述的青蒿素衍生物选自下组：蒿甲醚、二氢青蒿素、青蒿琥酯、双氢蒿甲醚、蒿乙醚等。 5.一种体外非治疗性地抑制血小板衍生生长因子受体A(PDGFRα)活性的方法，其特征在于，包括步骤：将青蒿素和/或青蒿素衍生物与细胞接触，从而抑制或拮抗细胞中血小板衍生生长因子受体A(PDGFRα)。 6.一种制剂组合的用途，所述制剂组合包括： (I)含青蒿素和/或青蒿素衍生物的制剂；和 (II)含吉西他宾或卡铂的制剂， 其特征在于，所述制剂组合用于制备： (a)抑制血小板衍生生长因子受体A的药物组合物或药盒；或 (b)预防和/或治疗肿瘤的药物组合物或药盒。 7.如权利要求6所述的用途，其特征在于，所述肿瘤为PDGFRα阳性肿瘤。 8.一种预防和/或治疗PDGFRα相关疾病的方法，其中所述疾病与PDGFRα的过表达或活性过高有关，其特征在于，包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物，或施用含青蒿素和/或青蒿素衍生物的药物组合物。 9.一种方法，所述方法用于体内或体外抑制PDGFRα酪氨酸激酶的活化、抑制PDGFRα的蛋白稳定性、促进PDGFRα泛素依赖的蛋白酶体介导的降解、抑制信号通路PI3K/AKT和MAPK/EPK的活化、和/或抑制PDGFRα阳性肿瘤细胞的生长、迁移和侵袭，其特征在于，所述方法包括步骤：给需要的对象施用青蒿素和/或青蒿素衍生物。 10.一种用于卵巢癌病理分级的试剂盒，其特征在于，所述试剂盒包含检测离体组织PDGFRα表达量的试剂。 Patent Citations (1) Publication numberPriority datePublication dateAssigneeTitle CN102010421A *2010-11-192011-04-13沈阳药科大学青蒿素类衍生物及其应用 Family To Family Citations * Cited by examiner, † Cited by third party Non-Patent Citations (6) Title YAOJIAO, ET. 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The invention discloses a kind of applications in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation.Qinghaosu has extensive biology and pharmacological action, and wherein effective antitumour effect causes extensive concern in recent years.The mechanism that qinghaosu plays specific anticancer activity is still unclear, this may limit its further development in preclinical and clinical setting.It is inhibited to hepatocellular carcinoma H22 that the present invention explores qinghaosu；And have found the mechanism of action of its anti-liver cancer and anti-, new method is provided for the treatment and diagnosis of liver cancer patient, is application based theoretical of the qinghaosu in clinical liver cancer treatment.Therefore, qinghaosu can be used as induction apoptosis of human hepatoma cell drug candidate and be researched and developed. 本发明公开了一种在制备治疗EMT激活的肝癌HepG2细胞候选药物中的应用。青蒿素具有广泛的生物和药理作用，其中有效的抗肿瘤作用近年来引起了广泛的关注。青蒿素发挥特定抗癌活性的机制仍不清楚，这可能限制其在临床前和临床环境中的进一步发展。本发明探索了青蒿素对肝癌细胞HepG2具有抑制作用；及发现了其抗肝癌的作用机制，为肝癌患者的治疗和诊断提供新方法，为青蒿素在临床肝癌治疗中的应用奠定理论基础。因此，青蒿素可作为诱导人肝癌细胞凋亡候选药物进行研究和开发。 Artemisinin is in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation Using Technical field The present invention relates to a kind of cancer treatment drugs, belong to biopharmaceutical technology, and in particular to a kind of qinghaosu exists Application in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation. Background technique One of the main reason for liver cancer (HCC) is global cancer related mortality, the liver cancer case more than 80% occur in The developing countries such as state and Africa.HCC has long latency, but while quickly growing usually to advanced stage is just diagnosed.These A possibility that feature is invaded plus its height causes the patient with the disease that cannot obtain the effective treatment.Non-operative treatment It is necessary, because the patient with big tumour (diameter > 5cm) or multiple lesions (> 3) is often unsuitable for hepatectomy.It is unfortunate , the activity of single chemotherapeutant is limited, with low-down treatment rate. Qinghaosu (ART) is a kind of natural products separated from plant ginghao (Artemesiaannua L), quilt It is widely used as anti-malaria medicaments.In recent years, ART and its derivative also have shown that with antitumaous effect, anticancer activity Main mechanism is to induce cell apoptosis.The selective anticancer potentiality and c-MYC, cdc25A, EGFR of qinghaosu and its derivative, The expression of the different moleculars such as gamma-glutamyl amine synzyme (GLCLR) is related.ART also plays in kinds cancer type and in vivo anti- Cancer effect. ART also adjusts the level of u-PA, MMP2, MMP7 and MMP9, all these all related to transfer.But qinghaosu is in liver Application and study on mechanism in cancer cell is still few, although detailed mechanism still needs to be illustrated. Summary of the invention Of the existing technology in order to solve the problems, such as, the purpose of the present invention is to provide a kind of biological therapy tumor candidate medicines Object qinghaosu is preparing the application in antitumor. The present invention is achieved through the following technical solutions: The present invention discloses a kind of application of qinghaosu in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation, Qinghaosu containing treatment effective dose pharmaceutically, the qinghaosu can be by inhibiting Beta- in hepatocellular carcinoma H22 Conversion of the catenin albuminous cell matter to cytoplasm, and then inhibit the generation of EMT, to inhibit the proliferation of HepG2. Application of the present invention, the qinghaosu be extracted from compound inflorescence plant Artemisia annua cauline leaf have peroxy A kind of colourless acicular crystal of the sesquiterpene lactone of group, molecular formula C15H22O5, belong to sesquiterpene lactone, preparing fusing point is 156- 157 DEG C, [a]D17=+66.3 ° (C=1.64 chloroform). Application of the present invention, concentration >=100 μM of the qinghaosu, the drug candidate can be made into tablet, system Preparation Method are as follows: 1) qinghaosu and starch are crossed into 80 meshes； 2) preparation of 10% starch slurry: 0.3g citric acid is dissolved in 20ml distilled water, and it is equal to add 2g starch dispersion Even, about 80 DEG C of heating makes starch gelatinization, uses after being cooled to temperature slurry； 3) fine powder of 30g qinghaosu is weighed in mortar, and 2g starch is added by several times and is ground for equivalent, is uniformly mixed, adds Enter appropriate amount of starch slurry and prepares softwood； 4) 20 mesh nylon mesh are prepared into wet granular, wet granular and 50~60 DEG C of oven drying 30min is carried out with 20 meshes Whole grain, particle is uniformly mixed with 1.5g talcum powder and 1g starch after whole grain, carries out punch die tabletting with 8mm punch die；Up to qinghaosu Tablet. Application of the present invention, the drug can be made into suspension injection, preparation method are as follows: 1) it takes oil for injection to be heated to 115~125 DEG C, keeps the temperature 1~2 hour, suspending agent is added, stirring and dissolving cools to 50 DEG C hereinafter, spare； 2) substance of step 1) is placed in colloid mills, 0.5~10.0g of qinghaosu is added, with the speed of 4000r/min Degree, suspension is made in high speed shear 30min, spare； 3) by the liquid of step 2), 0.1~5.0g of suspending agent, 0.05~0.5g of antioxidant, wetting agent are sequentially added 1.0~3.0g, flocculant 0.2~2.0g, it is stirring while adding, until substantially uniformity, then continuously grinding 10min, it is spare； 4) it by the liquid after the grinding of step 3), is placed in high pressure homogenizer, with pressure 50-55 megapascal, it is equal to carry out high pressure Matter, then, filling and sealing is packed to get 100mL sweet wormwood suspension injection. The invention has the advantages that and advantageous effects: 1) discovery qinghaosu is inhibited to human hepatoma HepG2 cell, and good time and concentration dependant is presented Property； 2) qinghaosu can induce the apoptosis of human hepatoma HepG2 cell； 3) qinghaosu can inhibit transhipment of the Beta-catenin from cytoplasm to nucleus, and then inhibit the generation of EMT, from And inhibit the invasion and transfer of tumour cell； The present invention provides scientific basis to develop new antitumor drug candidate, has important meaning to exploitation new Chinese medicine Justice. Detailed description of the invention Fig. 1 is that cell viability experiment detects qinghaosu to the Proliferation Ability of human hepatoma HepG2 cell in implementation column 1 of the present invention Effect. Fig. 2 is that cell clonal formation experiment detects qinghaosu to the proliferation of human hepatoma HepG2 cell in implementation column 1 of the present invention Inhibiting effect. Fig. 3 is the work that cell streaming experiment detection qinghaosu induces human hepatoma HepG2 cell's apoptosis in implementation column 2 of the present invention With. Fig. 4 is the expression that Western blot detects qinghaosu PARP albumen intracellular to HepG2 in implementation column 3 of the present invention Amount. Fig. 5 is that Western blot detects qinghaosu to Beta-catenin in HepG2 cytoplasm in implementation column 4 of the present invention The influence of albumen. Specific embodiment Below with reference to specific implementation column and the chart technical solution that the present invention will be described in detail, but the present invention be not limited in Under embodiment. The present invention provides a kind of biological therapy tumor candidate drug artemisinin and is preparing the application in antitumor.It is specifically related to A kind of qinghaosu inhibits Beta-catenin albumen in cytoplasm to the conversion of nucleus and then hepatoma Hep G 2 cells is induced to wither It dies；Cell strain used in the experiment in vitro is source of people liver cancer cells (HepG2). The low host toxicity of qinghaosu is the major impetus for developing qinghaosu as anticancer agent.Therefore it is the inventors discovered that green Artemisin has antihepatocarcinoma effect in human hepatoma HepG2 cell, can be used as the potential value of anti-liver cancer and anti-drug candidate exploitation. The present invention is tested by cell viability, cell clonal formation is tested, the experiment of cell streaming, Western blot reality It tests, the researchs such as immunofluorescence experiment confirm that qinghaosu has the mechanism of action of induction hepatocellular carcinoma H22 apoptosis. Testing qinghaosu used below is purchase in sigma company, the U.S.. Various test apparatuses and reagent are commercial goods, are that can buy to obtain by commercial sources.Wherein, qinghaosu It is made by effective component extracting in Chinese traditional herbs artemisia annua, in sigma company, the U.S., product type 361593 is green for purchase Artemisin content is 98%, and cell strain used in experiment in vitro is source of people liver cancer cells (HepG2), derives from U.S. ATCC cell Library. Embodiment 1: Tablet, preparation method is made in qinghaosu drug are as follows: 1) qinghaosu and starch are crossed into 80 meshes； 2) preparation of 10% starch slurry: 0.3g citric acid is dissolved in 20ml distilled water, and it is equal to add 2g starch dispersion Even, about 80 DEG C of heating makes starch gelatinization, uses after being cooled to temperature slurry； 3) fine powder of 30g qinghaosu is weighed in mortar, and 2g starch is added by several times and is ground for equivalent, is uniformly mixed, adds Enter appropriate amount of starch slurry and prepares softwood； 4) 20 mesh nylon mesh are prepared into wet granular, wet granular and 50~60 DEG C of oven drying 30min is carried out with 20 meshes Whole grain, particle is uniformly mixed with 1.5g talcum powder and 1g starch after whole grain, carries out punch die tabletting with 8mm punch die；Up to qinghaosu Tablet. Medication: Adult usual amounts: oral administration first takes 1g, takes within 6~8 hours 0.5g again, respectively takes within the 2nd, 3 0.5g, and the course for the treatment of 3 days, Total amount is 2.5g.Children 15mg/kg takes in 3 days according to the above method. Points for attention: First Trimester is used with caution. Embodiment 2: Suspension injection, preparation method is made in qinghaosu drug are as follows: 1) it takes oil for injection to be heated to 115~125 DEG C, keeps the temperature 1~2 hour, suspending agent is added, stirring and dissolving cools to 50 DEG C hereinafter, spare； 2) substance of step 1) is placed in colloid mills, 0.5~10.0g of qinghaosu is added, with the speed of 4000r/min Degree, suspension is made in high speed shear 30min, spare； 3) by the liquid of step 2), 0.1~5.0g of suspending agent, 0.05~0.5g of antioxidant, wetting agent 1.0 are sequentially added ~3.0g, flocculant 0.2~2.0g, it is stirring while adding, until substantially uniformity, then continuously grinding 10min, it is spare； 4) it by the liquid after the grinding of step 3), is placed in high pressure homogenizer, with pressure 50-55 megapascal, it is equal to carry out high pressure Matter, then, filling and sealing is packed to get 100mL sweet wormwood suspension injection. Medication: deep intramuscular injection: the 1st 200mg gives 100mg, each intramuscular injection 100mg on the the 2nd, 3 again after 6~8 hours, Accumulated dose 500mg (gives 100mg in other severe the 4th day) again.It is used in conjunction 3, daily intramuscular injection 300mg, total amount 900mg.Children 15mg/ Kg has been infused in 3 days according to the above method. Points for attention: First Trimester is used with caution. Compliance test result test example Test example 1-1 (inhibited proliferation of the qinghaosu to human hepatoma HepG2 cell) 1, Cell Viability Assay method measures cell Proliferation 1) it takes in logarithmic growth phase, the good HepG2 cell of growth conditions, it is (green containing 10%FBS and 1% with DMEM Mycin, 1% streptomysin) culture medium adjusts cell density to 3*104A/ml, is inoculated into 96 orifice plates, and every 100 μ L cell of hole is outstanding Liquid, while blank group is set, 37 DEG C, 5%CO2Overnight incubation. 2) after cell adherent growth well for 24 hours after, abandon old culture solution, the sweet wormwood diluted with DMEM culture medium be added Element, concentration are followed successively by 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 6 repetitions are arranged in every kind of concentration.Negative control group adds equivalent DMEM.Be put into incubator cultivate respectively for 24 hours, 48h and 72h. 3) after drug effect to corresponding time point, 96 orifice plates are taken out from incubator, are separately added into 20 μ LCellTiter-Luminescent Cell Viability Assay, pays attention to being protected from light, and is put into incubator and is incubated for 10 points Zhong Hou uses the absorbance of Bio-Rad cell town and country detector measurement Luminescent. 4) cell survival rate (cell viability%)=(dosing group/negative control group) × 100%. Experiment in triplicate, calculates separately under different time qinghaosu to the inhibition situation of human hepatoma HepG2 cell. Test example 1-2(cell clonal formation experiment) 1) it prepares cell suspension: abandoning old culture medium, after PBS washes one time plus the pancreatin of 1mL 0.25% digests 3min, micro- When microscopic observation most cells are rounded, abandon pancreatin, add 2 times added by the complete medium of pancreatin amount terminate digestion, will be thin in bottle Born of the same parents gently blow down, and are transferred in 15mL centrifuge tube, and 1000rpm is centrifuged 5min. 2) supernatant is abandoned, fresh culture is added, 1/3 cell suspension is reserved seed for planting, remaining is spare. 3) cell count and bed board: after cell suspension piping and druming mixes, 10 μ L are drawn, blood counting chamber is added under the microscope It counts.The adjusted cell suspension of 2ml concentration is added to every hole of 12 orifice plates, cell number is 2000/hole, 37 DEG C are placed in, 5%CO2It is cultivated in incubator, keeps it adherent overnight. 4) dosing: discarding old culture medium, and the qinghaosu diluted with DMEM culture medium is added, and concentration is followed successively by 0 μM, 50 μ M, 3 repetitions are arranged in 100 μM, 150 μM, every kind of concentration. 5) by plate at 37 DEG C, 5%CO2It is lower to be incubated for 7 days to allow bacterium colony to grow.During prolonged incubation, every three days more Change the fresh complete medium of the qinghaosu (0 μM, 50 μM, 100 μM, 150 μM) containing various concentration.It is washed carefully with 1X PBS Twice, 0.01% crystal violet solution dyes born of the same parents.Gel Doc TM XR+Imager (Bio-Rad) captures image.In order to quantify to collect The rate to be formed is fallen, the staining cell of colony form is dissolved in 10% (v/v) acetic acid, the quantitative absorbance at 540nm. 6) data statistics: Cell colonies assay=(dosing group/negative control group) × 100%. Cell viability is as the result is shown: act on respectively for 24 hours, after 48h, 72h, qinghaosu energy dose-dependant and time dependence suppression The proliferation of HepG2 cell processed. By Figure 1A as it can be seen that the drug concentration in same action time is higher, the cell proliferation inhibition rate compared with blank control Also with increasing. By Figure 1B as it can be seen that when qinghaosu concentration is 100 μM, to the variant statistics of the inhibition of human hepatoma HepG2 cell Meaning. Cell clonal formation experimental result is shown: compared with blank control group, and qinghaosu increases human hepatoma HepG2 cell It grows and significantly inhibits.And when qinghaosu concentration is 100 μM, the Colony formings of liver cancer cells inhibit 50% with Under, and as qinghaosu concentration increases, inhibitory effect is better.Therefore subsequent experimental we choose qinghaosu concentration be 100 μM.Such as Shown in Fig. 2. Test example 2 The influence of cell streaming experiment detection qinghaosu induction human hepatoma HepG2 cell's apoptosis. 1) it takes in logarithmic growth phase, the good HepG2 cell of growth conditions, it is (green containing 10%FBS and 1% with DMEM Mycin, 1% streptomysin) culture medium adjusts cell density to 5*106A/ml, is inoculated into 6 orifice plates, every 100 μ L cell suspension of hole, While blank group is set, and 37 DEG C, 5%CO2Overnight incubation. 2) after cell adherent growth well for 24 hours after, abandon old culture solution, the sweet wormwood diluted with DMEM culture medium be added Element, concentration are followed successively by 0 μM, 100 μM, and 3 repetitions are arranged in every kind of concentration.Negative control group adds the DMEM of equivalent.It is put into incubator Culture is for 24 hours. 3) cell culture fluid is sucked out to a suitable centrifuge tube, PBS washing attached cell is primary, and it is thin that appropriate pancreatin is added Born of the same parents' digestive juice (EDTA can be contained) vitellophag.Incubation at room temperature is absorbed to when gently piping and druming can be such that attached cell blows and beats Pancreatin cell dissociation buffer.It need to avoid the excessive digestion of pancreatin. 4) cell culture fluid collected in step 2a is added, slightly mixes, is transferred in centrifuge tube, 1000g is centrifuged 5 minutes, Supernatant is abandoned, cell is collected, cell is gently resuspended with PBS and counts.Note: the cell culture fluid being added in step 1 on the one hand can To collect the cell that apoptosis or necrosis occurs to have suspended, the serum in another aspect cell culture fluid can effectively inhibit or Neutralize remaining pancreatin；Remaining pancreatin can digest and the Annexin V-PE for subsequent addition of degrading causes dyeing to fail. 5) cell for taking 5-10 ten thousand to be resuspended, 1000g are centrifuged 5 minutes, abandon supernatant, and 195 μ l Annexin V-PE knot is added It closes liquid and cell is gently resuspended. 6) 5 μ l Annexin V-PE are added, mix gently.Note: due to the difference of cell sample, apoptosis and degree of necrosis Difference, can according to preliminary result optimize Annexin V-PE usage amount. 7) room temperature (20-25 DEG C) is protected from light incubation 10-20 minutes, is subsequently placed in ice bath.Aluminium foil can be used to be protected from light. Cell can be resuspended 2-3 times during incubation to improve and mark effect. flow cytomery is carried out immediately. 9) data statistics. The above experiment is in triplicate. Experimental result is shown: the bis- dyes of Annexin V-FITC/PI are the results show that be 100 μM of works in qinghaosu drug concentration With for 24 hours when, compared with blank control, the apoptosis rate of experimental group increases.The apoptosis rate of control group HepG2 cell is respectively 13.36%, after 100 μM of qinghaosu processing for 24 hours, the apoptosis rate of experimental group HepG2 cell is 24.16%, and apoptosis rate is obvious It increases (Fig. 3). Test example 3 The variation of Western blot detection qinghaosu PARP protein expression intracellular to HepG2. The GAP-associated protein GAP of investigation has: PARP. 1) it takes in logarithmic growth phase, the good HepG2 cell of growth conditions, it is (green containing 10%FBS and 1% with DMEM Mycin, 1% streptomysin) culture medium adjusts cell density to 5*106A/ml, is inoculated into 6 orifice plates, every 100 μ L cell suspension of hole, While blank group is set, and 37 DEG C, 5%CO2Overnight incubation. 2) after cell adherent growth well for 24 hours after, abandon old culture solution, the sweet wormwood diluted with DMEM culture medium be added Element, concentration are followed successively by 0 μM, 100 μM, 200 μM of every kind of concentration 3 repetitions of setting.Negative control group adds the DMEM of equivalent.It is put into Incubator culture is for 24 hours. 3) cell is taken out from incubator, sops up culture medium, and the PBS that ice is gently added is cleaned one time, discards residual liquid. 4) it is washed one time with PBS, cell is collected by centrifugation, tries one's best and exhausts supernatant, it is spare to leave cell precipitation. 5) it uses BCA kit to survey protein concentration: using BSA to do standard curve as standard specimen, 2 μ l are added in 96 orifice plates 37 DEG C of reaction 30min of protein solution and 200ul BCA working solution (A:B=50:1) detect absorbance in 450nm with microplate reader, Concentration of specimens is calculated using absorbance concentration calculation formula.It is reference by the minimum sample of wherein concentration, by all samples concentration It adjusts consistent. 6) v/2 albuminous degeneration loading Buffer 95 DEG C of denaturation 10min in metal bath are added, after centrifugation can it is direct on Sample or long-term preservation are in -80 DEG C of refrigerators. 7) loading: westernblotting loading, every hole loading 20ul select hole count, voltage according to sample size 200V, time 30min or so (pre-prepared colloid). transferring film: the filter paper of one piece of long 9cm, the pvdf membrane of wide 6cm and two same sizes are cut, film is put and is soaked in methyl alcohol Bubble, filter paper, film, gel and filter paper " sandwich " formula place it is wet turn 250mA, 90min, it is cathode that glue surface is surveyed to black one, is sure not Position is misplaced, bearing mark is carried out. 9) it closes: film being placed on after closing 1h in the milk of TBST preparation 5%, TBST is cleaned 4 times, each 5min. 10) be incubated for primary antibody: primary antibody is diluted with the Sodium azide 1:1000 of 5% BSA+0.02%.4 DEG C of shaking tables are incubated overnight, Primary antibody dilution can be placed on -20 DEG C of preservations and be used for multiple times. 11) it cleans: being cleaned 4 times with TBST, be placed on shaking table, low speed rocks, each 10min. 12) be incubated for secondary antibody: secondary antibody is diluted with 5% milk that TBST is prepared by 1:5000, is placed on shaking table, is incubated at room temperature 2h.Secondary antibody diluent had better not Reusability. 13) it cleans: rocking cleaning 4 times, each 5min using TBST. 14) expose: development liquid kit A and B liquid press 1:1 adapted, in use, covering film reaction 1min or so is put into egg It is exposed in white imaging system. The above experiment is in triplicate. As the result is shown: compared with blank control, after qinghaosu is added, the intracellular PARP protein expression up-regulation of HepG2 is poor Different statistically significant (P < 0.05), qinghaosu can cross induction HepG2 Apoptosis.As shown in Figure 4. Test example 4 4-1 qinghaosu influences the variation of Beta-catenin protein expression in HepG2 cytoplasm. Beta-catenin protein expression in 1.Western Blot experiment detection HepG2 cytoplasm 1) it takes in logarithmic growth phase, the good HepG2 cell of growth conditions, it is (green containing 10%FBS and 1% with DMEM Mycin, 1% streptomysin) culture medium adjusts cell density to 5*106A/ml, is inoculated into 6 orifice plates, every 100 μ L cell suspension of hole, While blank group is set, and 37 DEG C, 5%CO2Overnight incubation. 2) after cell adherent growth well for 24 hours after, abandon old culture solution, the sweet wormwood diluted with DMEM culture medium be added Element, concentration are followed successively by 0 μM, 100 μM, 200 μM of every kind of concentration 3 repetitions of setting.Negative control group adds the DMEM of equivalent.It is put into Incubator culture is for 24 hours. 3) cell is taken out from incubator, sops up culture medium, and the PBS that ice is gently added is cleaned one time, discards residual liquid. 4) 200 microlitres of suppressor proteins extraction agent A for being added to PMSF are added in every 20 microlitres of cell precipitations. 5) the violent Vortex of most high speed 5 seconds, completely suspends cell precipitation and scatter.If (cell precipitation is not complete It suspends and scatter entirely, the vortex time can be appropriately extended.) 6) ice bath 10-15 minutes. 7) B10 microlitres of suppressor proteins extraction agent is added.The violent Vortex of most high speed 5 seconds, ice bath 1 minute. the violent Vortex of most high speed 5 seconds, 4 DEG C of 12,000-16,000g are centrifuged 5 minutes. 9) supernatant is drawn immediately into the plastic tube of a pre-cooling, the suppressor proteins as extracted.It can make immediately With can also freeze.(precipitating must not be touched, the supernatant of minimum volume can be retained above precipitating, so as not to touch it is heavy It forms sediment.Obtainable supernatant after suppressor proteins extraction agent A cracking of every 2,000,000 cell in 200 microlitres of this products, cell Starching protein concentration is about 2-5mg/ml, and different cells are different.) 10) for precipitating, remaining supernatant is exhausted completely, and 50 microlitres of Nuclear extract extracting examinations for being added to PMSF are added Agent.(pollution of suppressor proteins can be brought by not exhausting supernatant.) 11) violent Vortex 15-30 seconds of most high speed completely suspend cell precipitation and scatter.Then ice bath is put back to In, violent Vortex 15-30 seconds, totally 30 minutes of high speed again every 1-2 minutes. 12) 4 DEG C of 12,000-16,000g are centrifuged 10 minutes. 13) supernatant is drawn immediately into the plastic tube of a pre-cooling, the Nuclear extract as extracted.It can make immediately With can also be frozen with -70 DEG C. 14) the RIPA cell pyrolysis liquid (containing protease inhibitors) of 100 μ l is added, scrapes cell with cell spatula, inhales Into EP pipe, it is inserted into ice and cracks 1h, during which overturn makes cell cracking abundant several times. 15) EP pipe is placed on 14000rpm in 4 DEG C of centrifuges and is centrifuged 20min, take supernatant. 16) it uses BCA kit to survey protein concentration: using BSA to do standard curve as standard specimen, 2 μ l are added in 96 orifice plates 37 DEG C of reaction 30min of protein solution and 200ul BCA working solution (A:B=50:1) detect absorbance in 450nm with microplate reader, Concentration of specimens is calculated using absorbance concentration calculation formula.It is reference by the minimum sample of wherein concentration, by all samples concentration It adjusts consistent. 17) v/2 albuminous degeneration loading Buffer 95 DEG C of denaturation 10min in metal bath are added, it can be direct after centrifugation Loading or long-term preservation are in -80 DEG C of refrigerators. 18) loading: western blotting loading, every hole loading 20ul select hole count, voltage according to sample size 200V, time 30min or so (pre-prepared colloid). 19) transferring film: the filter paper of one piece of long 9cm, the pvdf membrane of wide 6cm and two same sizes are cut, film is put in methyl alcohol Impregnate, filter paper, film, gel and filter paper " sandwich " formula place it is wet turn 250mA, 90min, it is cathode that glue surface is surveyed to black one, is cut Position is not misplaced, bearing mark is carried out. 20) it closes: film being placed on after closing 1h in the milk of TBST preparation 5%, TBST is cleaned 4 times, each 5min. 21) be incubated for primary antibody: primary antibody is diluted with the Sodium azide 1:1000 of 5% BSA+0.02%.4 DEG C of shaking tables are incubated overnight, Primary antibody dilution can be placed on -20 DEG C of preservations and be used for multiple times. 22) it cleans: being cleaned 4 times with TBST, be placed on shaking table, low speed rocks, each 10min. 23) be incubated for secondary antibody: secondary antibody is diluted with 5% milk that TBST is prepared by 1:5000, is placed on shaking table, is incubated at room temperature 2h.Secondary antibody diluent had better not Reusability. 24) it cleans: rocking cleaning 4 times, each 5min using TBST. 25) expose: development liquid kit A and B liquid press 1:1 adapted, in use, covering film reaction 1min or so is put into egg It is exposed in white imaging system. The above experiment is in triplicate. 4-2 immunofluorescence experiment detects Beta-catenin protein expression in HepG2 cytoplasm 1) it takes in logarithmic growth phase, the good HepG2 cell of growth conditions, it is (green containing 10%FBS and 1% with DMEM Mycin, 1% streptomysin) culture medium adjusts cell density to 5*106A/ml, is inoculated into 6 orifice plates, every 100 μ L cell suspension of hole, While blank group is set, and 37 DEG C, 5%CO2Overnight incubation. 2) after for 24 hours, renew fresh DMEM culture medium, the qinghaosu that concentration is 100 μM, control group addition etc. is added in experimental group The DMSO of amount, culture is for 24 hours. 3) liquid is blotted, one layer of 2-3mm diluted 4% formaldehyde of PBS is then covered on cell. 4) cell 15 minutes are fixed at room temperature. 5) fixer is blotted, three times with PBS rinsing, 5 minutes every time. 6) seal sheet glass 60 minutes in Block buffer. 7) when seal sheet glass, the middle dilution ratio recommended, prepares primary antibody in antibody dilution buffer to specifications. Block buffer is sucked, the primary antibody after dilution is added. 9) it is incubated overnight at a temperature of 4 DEG C. 10) three times with PBS rinsing, 5 minutes every time. 11) after the secondary antibody dilution of fluorescein will be coupled with antibody dilution buffer, it is small that it is protected from light incubation sample 1-2 at room temperature When. 12) secondary antibody Hoechst is protected from light incubation 1-2 hours, and PBS is washed 3 times, and 5 minutes every time, 5% glycerol mounting was burnt in copolymerization Microscopically observation.Experiment is in triplicate. As the result is shown: Western blot experimental result is shown, compared with blank control, after qinghaosu is added, in HepG2 cytoplasm The up-regulation of Beta-catenin protein expression, difference is statistically significant (P < 0.05), and trendless changes in nucleus, qinghaosu Transhipment of the Beta-catenin from cytoplasm to nucleus can enough be inhibited excessively, and then inhibit the generation of EMT, to inhibit tumour thin The invasion and transfer (as indicated by figures 5 a-5b) of born of the same parents.Immunofluorescence results are consistent (such as Fig. 5 C institute with Western blot result Show). The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, therefore Without departing from the technical solutions of the present invention, to the above embodiments according to the technical essence of the invention any simply to repair Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention. Claims (5) 1. application of the qinghaosu in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation, it is characterised in that: contain The qinghaosu for the treatment of effective dose pharmaceutically, the qinghaosu can be by inhibiting Beta- in hepatocellular carcinoma H22 Conversion of the catenin albuminous cell matter to cytoplasm, and then inhibit the generation of EMT, to inhibit the proliferation of HepG2. 2. application according to claim 1, it is characterised in that: the qinghaosu is from compound inflorescence plant Artemisia annua cauline leaf A kind of colourless acicular crystal of the sesquiterpene lactone for having peroxy-radical of middle extraction, molecular formula C15H22O5, belong to sesquiterpene lactone, Preparing fusing point is 156-157 DEG C, [a]D17=+66.3 ° (C=1.64 chloroform). 3. application according to claim 1, it is characterised in that: concentration >=100 μM of the qinghaosu. 4. application according to claim 1-3, it is characterised in that: the drug candidate can be made into tablet, system Preparation Method are as follows: 1) qinghaosu and starch are crossed into 80 meshes； 2) preparation of 10% starch slurry: 0.3g citric acid is dissolved in 20ml distilled water, and it is uniform to add 2g starch dispersion, is added About 80 DEG C of heat makes starch gelatinization, uses after being cooled to temperature slurry； 3) fine powder of 30g qinghaosu is weighed in mortar, and 2g starch is added by several times and is ground for equivalent, is uniformly mixed, and is added suitable Amount starch slurry prepares softwood； 4) 20 mesh nylon mesh are prepared into wet granular, wet granular and 50~60 DEG C of oven drying 30min is carried out whole with 20 meshes , particle is uniformly mixed with 1.5g talcum powder and 1g starch after whole grain, carries out punch die tabletting with 8mm punch die；Up to Artemisinin Tablets Agent. 5. application according to any one of claim 1-3, it is characterised in that: the drug can be made into suspension injection, Preparation method are as follows: 1) it takes oil for injection to be heated to 115~125 DEG C, keeps the temperature 1~2 hour, suspending agent is added, stirring and dissolving cools to 50 DEG C Hereinafter, spare； 2) substance of step 1) is placed in colloid mills, 0.5~l0.0g of qinghaosu is added, with the speed of 4000r/min, Suspension is made in high speed shear 30min, spare； 3) by the liquid of step 2), sequentially add 0.1~5.0g of suspending agent, 0.05~0.5g of antioxidant, wetting agent 1.0~ 3.0g, flocculant 0.2~2.0g, it is stirring while adding, until substantially uniformity, then continuously grinding 10min, it is spare； 4) it by the liquid after the grinding of step 3), is placed in high pressure homogenizer, with pressure 50-55 megapascal, progress is high-pressure homogeneous, so Afterwards, filling and sealing is packed to get 100mL sweet wormwood suspension injection. Patent Citations (1) Publication numberPriority datePublication dateAssigneeTitle CN101856352A *2009-04-102010-10-13中国科学院上海生命科学研究院Synergistic effect of arteannuim and derivative thereof on chemotherapeutic agent Family To Family Citations * Cited by examiner, † Cited by third party Non-Patent Citations (1) Title TAN WEIFENG等.: "Artemisinin inhibits in vitro and in vivo invasion and metastasis of human hepatocellular carcinomacells.", 《PHYTOMEDICINE》 * * Cited by examiner, † Cited by third party Similar Documents PublicationPublication DateTitle Abdel-Salam et al.2019Cytotoxicity of Luffa cylindrica (L.) M. 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And Related Applications Priority Applications (1) ApplicationPriority dateFiling dateTitle CN201910349857.7A2019-04-282019-04-28Application of the qinghaosu in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation Applications Claiming Priority (1) ApplicationFiling dateTitle CN201910349857.7A2019-04-28Application of the qinghaosu in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation https://patents.google.com/patent/CN110354118A/en 青蒿素在制备治疗EMT激活的肝癌HepG2细胞候选药物中的 应用 技术领域 本发明涉及一种肝癌治疗药物，属于生物制药技术领域，具体涉及一种青蒿素在制备治疗EMT激活的肝癌HepG2细胞候选药物中的应用。 背景技术 肝癌(HCC)是全球癌症相关死亡的主要原因之一，超过80％的肝癌病例发生在中国和非洲等发展中国家。HCC具有长潜伏期，但发展迅速通常到晚期时才被诊断出来。这些特征加上其高度入侵的可能性，导致患有该疾病的患者不能得到有效的治疗。非手术方法是必要的，因为患有大肿瘤(直径>5cm)或多个病灶(>3)的患者通常不适合肝切除术。不幸的是，单一化学治疗剂的活性是有限的，具有非常低的治疗率。 青蒿素(ART)是一种从植物青蒿(Artemesiaannua L)中分离出来的天然产物，被广泛用作抗疟疾药物。近年来，ART及其衍生物也已经显示出具有抗癌作用，其抗癌活性的主要机制是诱导细胞凋亡。青蒿素及其衍生物的选择性抗癌潜力与c-MYC， cdc25A，EGFR，γ-谷氨酰胺合成酶(GLCLR)等不同分子的表达有关。ART还在多种癌症类型及体内发挥抗癌作用。 ART还调节u-PA，MMP2，MMP7和MMP9的水平，所有这些都与转移相关。但青蒿素在肝癌细胞中的应用和作用机制研究尚少，虽然详细的机制仍有待阐明。 发明内容 为了解决现有技术存在的问题，本发明的目的在于提供一种生物治疗肿瘤候选药物青蒿素在制备抗肿瘤中的应用。 本发明是通过如下技术方案实现的： 本发明公开一种青蒿素在制备治疗EMT激活的肝癌HepG2细胞候选药物中的应用，含有药学上的治疗有效剂量的青蒿素，所述青蒿素能够通过抑制肝癌细胞HepG2 中Beta-catenin蛋白细胞质向细胞浆的转换，进而抑制EMT的发生，从而抑制HepG2 的增殖。 本发明所述的应用，所述青蒿素是从复合花序植物黄花蒿茎叶中提取的有过氧基团的倍半萜内酯的一种无色针状晶体，分子式为C15H22O5，属倍半萜内酯，制备熔点为 156-157℃，[a]D17＝+66.3°(C＝1.64氯仿)。 本发明所述的应用，所述青蒿素的浓度≥100μM，所述候选药物可制成片剂，其制备方法为： 1)将青蒿素和淀粉过80目筛； 2)10％淀粉浆的制备：将0.3g枸橼酸溶解于20ml蒸馏水中，再加入2g淀粉分散均匀，加热约80℃使淀粉糊化，冷却至温浆后使用； 3)称取30g青蒿素的细粉于乳钵中，等量分次加入2g淀粉进行研磨，混合均匀，加入适量淀粉浆制备软材； 4)将20目尼龙筛制备湿颗粒，将湿颗粒与50～60℃烘箱干燥30min，用20目筛进行整粒，整粒后颗粒与1.5g滑石粉和1g淀粉混合均匀，以8mm冲模进行冲模压片；即得青蒿素片剂。 本发明所述的应用，所述药物可制成混悬注射液，其制备方法为： 1)取注射用油加热至115～125℃，保温1～2小时，加入助悬剂，搅拌溶解，降温到50℃以下，备用； 2)将步骤1)的物质置于高速胶体磨中，加入青蒿素0.5～10.0g，以4000r/min的速度，高速剪切30min，制成混悬液，备用； 3)将步骤2)的液体，依次加入助悬剂0.1～5.0g、抗氧化剂0.05～0.5g、湿润剂1.0～3.0g、絮凝剂0.2～2.0g，边加边搅拌，直至完全均匀，再连续研磨10min，备用； 4)将步骤3)的研磨后的液体，置于高压均质机中，以压力50-55兆帕，进行高压均质，然后，灌装封口，包装，即得100mL青蒿混悬注射液。 本发明具有如下优点和有益技术效果： 1)发现青蒿素对人肝癌HepG2细胞具有抑制作用，并呈现良好的时间和浓度依赖性； 2)青蒿素可诱导人肝癌HepG2细胞的凋亡； 3)青蒿素可抑制Beta-catenin从细胞质向细胞核的转运，进而抑制EMT的发生，从而抑制肿瘤细胞的侵袭和转移； 本发明为研制新的抗肿瘤候选药物提供了科学依据，对开发中药新药具有重要意义。 附图说明 图1为本发明实施列1中细胞活力实验检测青蒿素对人肝癌HepG2细胞的增殖抑制作用。 图2为本发明实施列1中细胞克隆形成实验检测青蒿素对人肝癌HepG2细胞的增殖抑制作用。 图3为本发明实施列2中细胞流式实验检测青蒿素诱导人肝癌HepG2细胞凋亡的作用。 图4为本发明实施列3中Western blot检测青蒿素对HepG2细胞内PARP蛋白的表达量。 图5为本发明实施列4中Western blot检测青蒿素对HepG2细胞质内Beta-catenin蛋白的影响。 具体实施方式 下面结合具体实施列和图表详细说明本发明的技术方案，但本发明并不仅限于以下的实施例。 本发明提供一种生物治疗肿瘤候选药物青蒿素在制备抗肿瘤中的应用。具体涉及一种青蒿素抑制细胞质内Beta-catenin蛋白向细胞核的转换进而诱导肝癌HepG2细胞凋亡；所述的体外实验所用的细胞株为人源肝癌细胞(HepG2)。 青蒿素的低宿主毒性，是开发青蒿素作为抗癌剂的主要动力。故本发明人发现青蒿素在人肝癌HepG2细胞中具有抗肝癌作用，可以作为抗肝癌候选药物开发的潜在价值。 本发明通过细胞活力测试、细胞克隆形成实验、细胞流式实验、Western blot实验、免疫荧光实验等研究证实了青蒿素具有诱导肝癌细胞HepG2凋亡的作用机制。 以下实验所用的青蒿素是购买于美国sigma公司。 各种试验仪器与试剂均为市售商品，均为可通过商业途径购买获得。其中，青蒿素由传统中草药黄花蒿中提取有效成分制得，购买于美国sigma公司，产品型号为361593，青蒿素含量为98％，体外实验所用的细胞株为人源肝癌细胞(HepG2)，来源于美国ATCC 细胞库。 实施例1： 将青蒿素药物制成片剂，其制备方法为： 1)将青蒿素和淀粉过80目筛； 2)10％淀粉浆的制备：将0.3g枸橼酸溶解于20ml蒸馏水中，再加入2g淀粉分散均匀，加热约80℃使淀粉糊化，冷却至温浆后使用； 3)称取30g青蒿素的细粉于乳钵中，等量分次加入2g淀粉进行研磨，混合均匀，加入适量淀粉浆制备软材； 4)将20目尼龙筛制备湿颗粒，将湿颗粒与50～60℃烘箱干燥30min，用20目筛进行整粒，整粒后颗粒与1.5g滑石粉和1g淀粉混合均匀，以8mm冲模进行冲模压片；即得青蒿素片剂。 给药方法： 成人常用量：口服给药，先服1g，6～8小时再服0.5g，第2、3日各服0.5g，疗程3日，总量为2.5g。小儿15mg/kg，按上述方法3日内服完。 注意事项：妊娠早期慎用。 实施例2： 将青蒿素药物制成混悬注射液，其制备方法为： 1)取注射用油加热至115～125℃，保温1～2小时，加入助悬剂，搅拌溶解，降温到50℃以下，备用； 2)将步骤1)的物质置于高速胶体磨中，加入青蒿素0.5～10.0g，以4000r/min的速度，高速剪切30min，制成混悬液，备用； 3)将步骤2)的液体，依次加入助悬剂0.1～5.0g、抗氧化剂0.05～0.5g、湿润剂1.0～3.0g、絮凝剂0.2～2.0g，边加边搅拌，直至完全均匀，再连续研磨10min，备用； 4)将步骤3)的研磨后的液体，置于高压均质机中，以压力50-55兆帕，进行高压均质，然后，灌装封口，包装，即得100mL青蒿混悬注射液。 给药方法：深部肌注：第1次200mg，6～8小时后再给100mg，第2、3日各肌注100mg，总剂量500mg(别重症第4天再给100mg)。连用3日，每日肌注300mg，总量900mg。小儿15mg/kg，按上述方法3日内注完。 注意事项：妊娠早期慎用。 效果验证试验例 试验例1-1(青蒿素对人肝癌HepG2细胞的增殖抑制作用) 1、Cell Viability Assay法测定细胞增殖 1)取处于对数生长期，生长状态良好的HepG2细胞，用DMEM(含有10％FBS 和1％青霉素、1％链霉素)培养基调整细胞密度到3*104个/ml，接种到96孔板，每孔 100μL细胞悬液，同时设置空白组，37℃，5％CO2培养过夜。 2)待细胞贴壁生长良好24h后，弃旧的培养液，加入用DMEM培养基稀释过的青蒿素，浓度依次为0μM、50μM、100μM、150μM、200μM，每种浓度设置6个重复。阴性对照组加等量的DMEM。放入培养箱分别培养24h、48h和72h。 3)待药物作用到相应时间点后，从培养箱中取出96孔板，分别加入 20μLCellTiter-Luminescent Cell Viability Assay，注意避光，放入培养箱孵育10分钟后，使用Bio-Rad细胞城乡检测仪测量Luminescent的吸光度。 4)细胞存活率(cell viability％)＝(加药组/阴性对照组)×100％。 实验重复三次，分别计算不同时间下青蒿素对人肝癌HepG2细胞的抑制情况。 试验例1-2(细胞克隆形成实验) 1)制备细胞悬液：弃旧培养基，PBS洗一遍后加1mL 0.25％的胰酶消化3min，显微镜下观察大部分细胞变圆时，弃胰酶，加2倍所加胰酶量的完全培养基终止消化，将瓶内细胞轻轻吹下，并转移到15mL离心管中，1000rpm离心5min。 2)弃上清，加入新鲜培养基，1/3的细胞悬液留种，其余备用。 3)细胞计数和铺板：细胞悬液吹打混匀后，吸取10μL加入血球计数板在显微镜下计数。向12孔板的每孔加入2ml浓度调整过的细胞悬液，细胞数为2000个/孔，置于 37℃，5％CO2培养箱中培养，使其过夜贴壁。 4)加药：弃去旧培养基，加入用DMEM培养基稀释过的青蒿素，浓度依次为0μM、 50μM、100μM、150μM，每种浓度设置3个重复。 5)将平板在37℃，5％CO2下孵育7天以允许菌落生长。在长期孵育期间，每三天更换含有不同浓度的青蒿素(0μM、50μM、100μM、150μM)的新鲜完全培养基。用 1X PBS洗涤细胞两次，0.01％结晶紫溶液染色。Gel Doc TM XR+Imager(Bio-Rad)捕获图像。为了量化集落形成的速率，将集落形式的染色细胞溶解在10％(v/v)乙酸中，在540nm处定量吸光度。 6)数据统计：细胞克隆形成率＝(加药组/阴性对照组)×100％。 细胞活力结果显示：分别作用24h、48h、72h后，青蒿素能剂量依赖和时间依赖性抑制HepG2细胞的增殖。 由图1A可见，在同一作用时间的药物浓度越高，与空白对照相比细胞增殖抑制率也随着增高。 由图1B可见，在青蒿素浓度为100μM时，对人肝癌HepG2细胞的抑制有差异统计学意义。 细胞克隆形成实验结果显示：与空白对照组相比较，青蒿素对人肝癌HepG2细胞增殖具有明显的抑制作用。且在青蒿素浓度为100μM时，肝癌细胞的集落形成抑制在 50％以下，且随着青蒿素浓度增大，抑制效果越好。故后续实验我们选取青蒿素浓度为 100μM。如图2所示。 试验例2 细胞流式实验检测青蒿素诱导人肝癌HepG2细胞凋亡的影响。 1)取处于对数生长期，生长状态良好的HepG2细胞，用DMEM(含有10％FBS 和1％青霉素、1％链霉素)培养基调整细胞密度到5*106个/ml，接种到6孔板，每孔100μL 细胞悬液，同时设置空白组，37℃，5％CO2培养过夜。 2)待细胞贴壁生长良好24h后，弃旧的培养液，加入用DMEM培养基稀释过的青蒿素，浓度依次为0μM、100μM，每种浓度设置3个重复。阴性对照组加等量的DMEM。放入培养箱培养24h。 3)将细胞培养液吸出至一合适离心管内，PBS洗涤贴壁细胞一次，加入适量胰酶细胞消化液(可含有EDTA)消化细胞。室温孵育至轻轻吹打可以使贴壁细胞吹打下来时，吸除胰酶细胞消化液。需避免胰酶的过度消化。 4)加入步骤2a中收集的细胞培养液，稍混匀，转移到离心管内，1000g离心5分钟，弃上清，收集细胞，用PBS轻轻重悬细胞并计数。注意：加入步骤1中的细胞培养液一方面可以收集已经悬浮的发生凋亡或坏死的细胞，另一方面细胞培养液中的血清可以有效抑制或中和残留的胰酶；残留的胰酶会消化并降解后续加入的Annexin V-PE导致染色失败。 5)取5-10万重悬的细胞，1000g离心5分钟，弃上清，加入195μl Annexin V-PE 结合液轻轻重悬细胞。 6)加入5μl Annexin V-PE，轻轻混匀。注：由于细胞样品的差异、凋亡和坏死程度的差异，可根据预实验结果优化Annexin V-PE的使用量。 7)室温(20-25℃)避光孵育10-20分钟，随后置于冰浴中。可以使用铝箔进行避光。孵育过程中可以重悬细胞2-3次以改善标记效果。 8)随即进行流式细胞仪检测。 9)数据统计。 以上实验重复三次。 实验结果显示：Annexin V-FITC/PI双染结果显示，在青蒿素药物浓度为100μM作用24h时，与空白对照相比较，实验组的细胞凋亡率增加。对照组HepG2细胞的凋亡率分别为13.36％，经100μM青蒿素处理24h后，实验组HepG2细胞的凋亡率为24.16％，其凋亡率明显升高(图3)。 试验例3 Western blot检测青蒿素对HepG2细胞内PARP蛋白表达的变化。 考察的相关蛋白有：PARP。 1)取处于对数生长期，生长状态良好的HepG2细胞，用DMEM(含有10％FBS 和1％青霉素、1％链霉素)培养基调整细胞密度到5*106个/ml，接种到6孔板，每孔100μL 细胞悬液，同时设置空白组，37℃，5％CO2培养过夜。 2)待细胞贴壁生长良好24h后，弃旧的培养液，加入用DMEM培养基稀释过的青蒿素，浓度依次为0μM、100μM、200μM每种浓度设置3个重复。阴性对照组加等量的 DMEM。放入培养箱培养24h。 3)从培养箱中取出细胞，吸掉培养基，轻轻加入冰的PBS清洗一遍，弃掉残留液体。 4)用PBS洗一遍，离心收集细胞，尽最大努力吸尽上清，留下细胞沉淀备用。 5)使用BCA试剂盒测蛋白浓度：用BSA作为标样做标准曲线，在96孔板中加入 2μl蛋白溶液和200ul BCA工作液(A：B＝50：1)37℃反应30min，用酶标仪在450nm 检测吸光度，利用吸光度浓度计算公式计算样本浓度。按其中浓度最低的样品为参照，将所有样品浓度调节一致。 6)加入v/2蛋白变性loading Buffer在金属浴中95℃变性10min，离心后可直接上样，或长期保存在-80℃冰箱。 7)上样：westernblotting上样，每孔上样20ul，根据样品数量选择孔数，电压200V，时间30min左右(预制胶)。 8)转膜：切一块长9cm、宽6cm的PVDF膜和两张相同大小的滤纸，将膜放在甲醇中浸泡，滤纸、膜、凝胶和滤纸“三明治”式放置湿转250mA、90min，胶面向黑的一测即负极，切勿放错位置，做好方向标记。 9)封闭：将膜放在TBST配制5％的牛奶中封闭1h后，TBST清洗4次，每次5min。 10)孵育一抗：一抗用5％的BSA+0.02％的叠氮钠1：1000稀释。4℃摇床孵育过夜，一抗稀释液可有放在-20℃保存多次使用。 11)清洗：用TBST清洗4次，放在摇床，低速摇晃，每次10min。 12)孵育二抗：二抗用TBST配制的5％牛奶按1：5000稀释，放置在摇床，室温孵育2h。二抗稀释液最好不要反复使用。 13)清洗：使用TBST摇晃清洗4次，每次5min。 14)曝光：显影液试剂盒A与B液按1：1配用，使用时，覆盖住膜反应1min左右放入蛋白成像系统中曝光。 以上实验重复三次。 结果显示：与空白对照相比较，加入青蒿素后，HepG2细胞内PARP蛋白表达上调，差异有统计学意义(P<0.05)，青蒿素能过诱导HepG2细胞凋亡。如图4所示。 试验例4 4-1青蒿素对HepG2细胞质内Beta-catenin蛋白表达的变化影响。 1.Western Blot实验检测HepG2细胞质内Beta-catenin蛋白表达 1)取处于对数生长期，生长状态良好的HepG2细胞，用DMEM(含有10％FBS 和1％青霉素、1％链霉素)培养基调整细胞密度到5*106个/ml，接种到6孔板，每孔100μL 细胞悬液，同时设置空白组，37℃，5％CO2培养过夜。 2)待细胞贴壁生长良好24h后，弃旧的培养液，加入用DMEM培养基稀释过的青蒿素，浓度依次为0μM、100μM、200μM每种浓度设置3个重复。阴性对照组加等量的 DMEM。放入培养箱培养24h。 3)从培养箱中取出细胞，吸掉培养基，轻轻加入冰的PBS清洗一遍，弃掉残留液体。 4)每20微升细胞沉淀加入200微升添加了PMSF的细胞浆蛋白抽提试剂A。 5)最高速剧烈Vortex 5秒，把细胞沉淀完全悬浮并分散开。(如果细胞沉淀没有完全悬浮并分散开，可以适当延长vortex时间。) 6)冰浴10-15分钟。 7)加入细胞浆蛋白抽提试剂B10微升。最高速剧烈Vortex 5秒，冰浴1分钟。 8)最高速剧烈Vortex 5秒，4℃12,000-16,000g离心5分钟。 9)立即吸取上清至一预冷的塑料管中，即为抽提得到的细胞浆蛋白。可以立即使用，也可以冻存。(千万不要触及沉淀，可以在沉淀上方保留极小体积的上清，以免触及沉淀。每200万细胞用200微升本产品中的细胞浆蛋白抽提试剂A裂解后可获得的上清，其细胞浆蛋白浓度约为2-5mg/ml，不同细胞有所不同。) 10)对于沉淀，完全吸尽残余的上清，加入50微升添加了PMSF的细胞核蛋白抽提试剂。(不吸尽上清会带来细胞浆蛋白的污染。) 11)最高速剧烈Vortex 15-30秒，把细胞沉淀完全悬浮并分散开。然后放回冰浴中，每隔1-2分钟再高速剧烈Vortex 15-30秒，共30分钟。 12)4℃12,000-16,000g离心10分钟。 13)立即吸取上清至一预冷的塑料管中，即为抽提得到的细胞核蛋白。可以立即使用，也可以-70℃冻存。 14)加入100μl的RIPA细胞裂解液(含有蛋白酶抑制剂)，用细胞刮铲刮下细胞，吸到EP管中，插入冰里裂解1h，期间颠倒几次使细胞裂解充分。 15)将EP管放在4℃离心机中14000rpm离心20min，取上清。 16)使用BCA试剂盒测蛋白浓度：用BSA作为标样做标准曲线，在96孔板中加入2μl蛋白溶液和200ul BCA工作液(A:B＝50：1)37℃反应30min，用酶标仪在450nm 检测吸光度，利用吸光度浓度计算公式计算样本浓度。按其中浓度最低的样品为参照，将所有样品浓度调节一致。 17)加入v/2蛋白变性loading Buffer在金属浴中95℃变性10min，离心后可直接上样，或长期保存在-80℃冰箱。 18)上样：western blotting上样，每孔上样20ul，根据样品数量选择孔数，电压200V，时间30min左右(预制胶)。 19)转膜：切一块长9cm、宽6cm的PVDF膜和两张相同大小的滤纸，将膜放在甲醇中浸泡，滤纸、膜、凝胶和滤纸“三明治”式放置湿转250mA、90min，胶面向黑的一测即负极，切勿放错位置，做好方向标记。 20)封闭：将膜放在TBST配制5％的牛奶中封闭1h后，TBST清洗4次，每次5min。 21)孵育一抗：一抗用5％的BSA+0.02％的叠氮钠1：1000稀释。4℃摇床孵育过夜，一抗稀释液可有放在-20℃保存多次使用。 22)清洗：用TBST清洗4次，放在摇床，低速摇晃，每次10min。 23)孵育二抗：二抗用TBST配制的5％牛奶按1：5000稀释，放置在摇床，室温孵育2h。二抗稀释液最好不要反复使用。 24)清洗：使用TBST摇晃清洗4次，每次5min。 25)曝光：显影液试剂盒A与B液按1：1配用，使用时，覆盖住膜反应1min左右放入蛋白成像系统中曝光。 以上实验重复三次。 4-2免疫荧光实验检测HepG2细胞质内Beta-catenin蛋白表达 1)取处于对数生长期，生长状态良好的HepG2细胞，用DMEM(含有10％FBS 和1％青霉素、1％链霉素)培养基调整细胞密度到5*106个/ml，接种到6孔板，每孔100μL 细胞悬液，同时设置空白组，37℃，5％CO2培养过夜。 2)24h后，换新鲜的DMEM培养基，实验组加入浓度为100μM的青蒿素，对照组加入等量的DMSO，培养24h。 3)吸干液体，随后在细胞上覆盖一层2-3mm用PBS稀释的4％甲醛。 4)室温下固定细胞15分钟。 5)吸干固定液，用PBS漂洗三次，每次5分钟。 6)在封闭缓冲液中封闭标本60分钟。 7)封闭标本时，按照说明书中推荐的稀释比例，在抗体稀释缓冲液中配制一抗。 8)吸去封闭缓冲液，加入稀释后的一抗。 9)4℃温度下孵育过夜。 10)用PBS漂洗三次，每次5分钟。 11)用抗体稀释缓冲液将偶联荧光素的二抗稀释后，室温下避光孵育标本1-2小时。 12)二抗Hoechst避光孵育1-2小时，PBS洗3遍,每次5分钟，5％甘油封片，在共聚焦显微镜下观察。实验重复三次。 结果显示： Western blot实验结果显示，与空白对照相比较，加入青蒿素后，HepG2细胞质内Beta-catenin蛋白表达上调，差异有统计学意义(P<0.05)，而细胞核内无趋势变化，青蒿素能过够抑制Beta-catenin从细胞质向细胞核的转运，进而抑制EMT的发生，从而抑制肿瘤细胞的侵袭和转移(如图5A-5B所示)。免疫荧光结果与Western blot结果相符合(如图5C所示)。 以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，故凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。 Claims (5) 1.青蒿素在制备治疗EMT激活的肝癌HepG2细胞候选药物中的应用，其特征在于：含有药学上的治疗有效剂量的青蒿素，所述青蒿素能够通过抑制肝癌细胞HepG2中Beta-catenin蛋白细胞质向细胞浆的转换，进而抑制EMT的发生，从而抑制HepG2的增殖。 2.根据权利要求1所述的应用，其特征在于：所述青蒿素是从复合花序植物黄花蒿茎叶中提取的有过氧基团的倍半萜内酯的一种无色针状晶体，分子式为C15H22O5，属倍半萜内酯，制备熔点为156-157℃，[a]D17＝+66.3°(C＝1.64氯仿)。 3.根据权利要求1所述的应用，其特征在于：所述青蒿素的浓度≥100μM。 4.根据权利要求1-3任一项所述的应用，其特征在于：所述候选药物可制成片剂，其制备方法为： 1)将青蒿素和淀粉过80目筛； 2)10％淀粉浆的制备：将0.3g枸橼酸溶解于20ml蒸馏水中，再加入2g淀粉分散均匀，加热约80℃使淀粉糊化，冷却至温浆后使用； 3)称取30g青蒿素的细粉于乳钵中，等量分次加入2g淀粉进行研磨，混合均匀，加入适量淀粉浆制备软材； 4)将20目尼龙筛制备湿颗粒，将湿颗粒与50～60℃烘箱干燥30min，用20目筛进行整粒，整粒后颗粒与1.5g滑石粉和1g淀粉混合均匀，以8mm冲模进行冲模压片；即得青蒿素片剂。 5.根据权利要求1-3中任一项所述的应用，其特征在于：所述药物可制成混悬注射液，其制备方法为： 1)取注射用油加热至115～125℃，保温1～2小时，加入助悬剂，搅拌溶解，降温到50℃以下，备用； 2)将步骤1)的物质置于高速胶体磨中，加入青蒿素0.5～l0.0g，以4000r/min的速度，高速剪切30min，制成混悬液，备用； 3)将步骤2)的液体，依次加入助悬剂0.1～5.0g、抗氧化剂0.05～0.5g、湿润剂1.0～3.0g、絮凝剂0.2～2.0g，边加边搅拌，直至完全均匀，再连续研磨10min，备用； 4)将步骤3)的研磨后的液体，置于高压均质机中，以压力50-55兆帕，进行高压均质，然后，灌装封口，包装，即得100mL青蒿混悬注射液。 Patent Citations (1) Publication numberPriority datePublication dateAssigneeTitle CN101856352A *2009-04-102010-10-13中国科学院上海生命科学研究院青蒿素及其衍生物对化疗剂的协同作用 Family To Family Citations * Cited by examiner, † Cited by third party Non-Patent Citations (1) Title TAN WEIFENG等.: "Artemisinin inhibits in vitro and in vivo invasion and metastasis of human hepatocellular carcinomacells.", 《PHYTOMEDICINE》 * * Cited by examiner, † Cited by third party Similar Documents 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The invention belongs to biomedicine technical fields, specifically, being related to a kind of combination product and its application for preventing and/or treating cerebral ischemia.The combination product is the pharmaceutical composition containing Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound；Or the combination for the first medicament containing Sur1 inhibitor and the second medicament containing qinghaosu Sesquiterpene lactones compound.Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound are formed combination product by the present invention in appropriate proportions, has the function of synergy, than being applied alone Sur1 inhibitor or qinghaosu Sesquiterpene lactones compound to have better therapeutic effect to cerebral ischemia diseases. 本发明属于生物医药技术领域，具体地说，涉及一种用于预防和/或治疗脑缺血的组合产品及其应用。所述的组合产品为含有Sur1抑制剂和青蒿素倍半萜内酯类化合物的药物组合物；或为含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂的组合。本发明将Sur1抑制剂和青蒿素倍半萜内酯类化合物以适当比例形成组合产品，具有协同增效的作用，比单用Sur1抑制剂或者青蒿素倍半萜内酯类化合物对脑缺血性疾病有更好的治疗效果。 A kind of combination product and its application for preventing and/or treating cerebral ischemia Technical field The invention belongs to biomedicine technical fields, specifically, being related to a kind of for preventing and/or treating cerebral ischemia Combination product and its application. Background technology Cerebral arterial thrombosis has high incidence, high mortality, high disability rate, high relapse rate, the several spies of high health care costs Point is the great chronic disease for endangering China's residents ' health.Clinical data statistics display, the global cerebral apoplexy death rate already exceed cancer Disease becomes the highest disease of the Single diseases death rate.It is well known that inflammatory reaction is the important set during cerebral ischemia pathology damage At part, and aggravate the latency of cerebral ischemia.In addition to this, cerebral arterial thrombosis is often accompanied by brain edema and hemorrhagic Conversion, wherein brain edema is the complication of cerebral arterial thrombosis most serious, and 80% dies of the palsy later stage in apoplexy patient death Pernicious brain edema.Therefore the prevention of inflammation and brain edema is significant to the treatment of cerebral arterial thrombosis. Sulfonylureas receptor 1 (sulfonylurea receptor 1, Sur1)-transient receptor potential channel M4 types The channel (transient receptor potential melastatin 4, Trpm4) plays the formation of brain edema important Effect.Pertinent literature reports, cerebral arterial thrombosis occur be within 4-8 hour brain edema formation critical period, in this period ischemic The Sur1 receptors in the position channels cerebrovascular tapetum cell film Sur1-Trpm4 will appear the expression of irritability height, the Sur1 of great expression Receptor causes the channels Sur1-Trpm4 to be activated.The activation in the channels Sur1-Trpm4 increases cationic especially sodium ion and enters cell It is interior, since the reason driving water of osmotic pressure pours in into the cell, causes ishemic part cell or even tissue that oedema occurs, be soldier There is the key pathological mechanism of brain edema in middle patient.Sur1 inhibitor is combined the opening for inhibiting this channel with Sur1 receptors, from And effectively inhibit the formation of brain edema.Representative Sur1 inhibitor glibenclamide is directed to the treatment of palsy associated with hydrocephalus in U.S. State enters the clinical III phases. Qinghaosu and its derivative are a kind of new antimalarial agents of China scientific worker research and development, are now widely used for The treatment of malaria.With the continuous deepening of research, other than its Antimalarial, scientist is also found that qinghaosu and its spreads out in succession Biology pharmacological action in other respects, including antitumor action, the effect to immunocyte, anti-fibrosis effect, it is anti-it is fat, Anti-diabetic and antifungic action etc., wherein Antimalarial and antitumor action are the most noticeable.Qinghaosu and its derivative Research of the object in terms of inflammatory is based primarily upon its therapeutic effect to autoimmune disease, such as lupus erythematosus and rheumatoid arthrosis Inflammation, researches show that it includes NF-Kb and cell factor, such as IL-1, IL-6 and IL-8 etc. equal to multiple inflammatory relevant target proteins There is regulating and controlling effect.But the application of qinghaosu and its derivative in terms for the treatment of cerebral ischemia diseases so far there are no research report. In view of this special to propose the present invention. Invention content The technical problem to be solved in the present invention is to overcome the deficiencies of the prior art and provide a kind of for preventing and/or controlling Combination product and its application of cerebral ischemia are treated, has synergistic effect between the ingredient of the combination product. In order to solve the above technical problems, the present invention adopts the following technical scheme that： A kind of combination product for preventing and/or treating cerebral ischemia, wherein the combination product is to press down containing Sur1 The pharmaceutical composition of preparation and qinghaosu Sesquiterpene lactones compound； Or it is the first medicament containing Sur1 inhibitor and the second medicament containing qinghaosu Sesquiterpene lactones compound Combination. Specifically, on the one hand, it is provided by the present invention for preventing and/or treating the combination product of cerebral ischemia, it can be with It is that Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound are put together and to form a kind of pharmaceutical composition, the pharmaceutical composition Can a kind of compound preparation further be made with pharmaceutically acceptable auxiliary material in object. On the other hand, provided by the present invention for preventing and/or treat the combination product of cerebral ischemia, can also be by containing There are the first medicament of Sur1 inhibitor and the second medicament containing qinghaosu Sesquiterpene lactones compound to carry out drug combination. When drug combination, the medication of the first medicament and second medicament sequence in no particular order, can be first with the first medicament, can also be first with the Two medicaments can simultaneously be used with two medicaments. Further, in the pharmaceutical composition Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound quality Than for (1~3):(2500~5000)； Qinghaosu Sesquiterpene lactones compound in Sur1 inhibitor and second medicament in first medicament in the combination Mass ratio be (1~3):(2500~5000). Further, the pharmaceutical composition is Sur1 inhibitor, qinghaosu Sesquiterpene lactones compound and pharmacy Pharmaceutically acceptable dosage form, optimizing injection, more preferable injection or freeze-dried powder is made in upper acceptable auxiliary material. Its supplementary product kind and preparation method can refer to the prior art. Further, first medicament is made with pharmaceutically acceptable auxiliary material for Sur1 inhibitor and can pharmaceutically connect The dosage form received； The second medicament is that pharmacy is made with pharmaceutically acceptable auxiliary material in qinghaosu Sesquiterpene lactones compound Upper acceptable dosage form, optimizing injection, more preferable injection or freeze-dried powder. In the present invention, by the first medicament containing Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound can be contained Second medicament carry out drug combination.First medicament or second medicament can be existing preparations in the prior art, Can be that Sur1 inhibitor or qinghaosu Sesquiterpene lactones compound are added pharmaceutically acceptable auxiliary material respectively to make respectively At pharmaceutically acceptable dosage form.The preparation method of supplementary product kind and each dosage form can refer to the prior art. Further, the Sur1 inhibitor is selected from glibenclamide, orinase, Repaglinide, meglitinide, miaow One or more of lattice row azoles or Glimepiride. Further, the qinghaosu Sesquiterpene lactones compound be the sesquiterpene lactone extracted from artemisia annua and The derivative of qinghaosu. Further, the qinghaosu Sesquiterpene lactones compound is Artesunate, dihydroartemisinine or Artemether. The present invention also provides the combination products to prepare the application in treating Imaging in Patients with Cerebral Ischemia Disease drug. The cerebral ischemia is cerebral apoplexy. After adopting the above technical scheme, the present invention has the advantages that compared with prior art： The present invention provides a kind of containing Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound, wherein Sur1 inhibitor Can be by inhibiting sur1 to mitigate brain edema, and qinghaosu Sesquiterpene lactones compound can be by inhibiting inflammatory factor and related egg White hair waves anti-inflammatory effect.The two has the function of synergy when being used cooperatively in appropriate proportions.By Sur1 inhibitor and sweet wormwood Plain Sesquiterpene lactones compound drug combination in appropriate proportions, can be than being applied alone in Sur1 inhibitor or qinghaosu sequiterpene Ester type compound has better therapeutic effect to cerebral ischemia diseases. The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings. Description of the drawings Fig. 1 shows cerebral infarction volume percentage, sham-operation group vs model groups,###P ＜ 0.001；Administration group vs model groups,**P ＜ 0.01,*P ＜ 0.05； Fig. 2 shows edema volume percentage, sham-operation group vs model groups,###p<0.001；Administration group vs model groups,**P ＜ 0.01； Fig. 3 shows Serum TNF-α and IL-6 expressions, sham-operation group vs model groups,###P ＜ 0.001；Administration group vs models Group,***P ＜ 0.001,*P ＜ 0.05； It should be noted that these attached drawings and verbal description are not intended to the design model limiting the invention in any way It encloses, but is that those skilled in the art illustrate idea of the invention by referring to specific embodiments. Specific implementation mode In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention In attached drawing, the technical solution in embodiment is clearly and completely described, following embodiment for illustrating the present invention, but It is not limited to the scope of the present invention. It is below embodiment 1 to embodiment 7, the composition of pharmaceutical composition is shown in Table 1 in each embodiment, preparation method To weigh each substance by the quality, then it is mixed uniformly to get pharmaceutical composition. Table 1 Aforementioned pharmaceutical compositions can further add pharmaceutically acceptable auxiliary material and be prepared into pharmaceutically acceptable dosage form, Optimizing injection, including but not limited to injection or freeze-dried powder. The research of test example 1, glibenclamide and Artesunate to cerebral ischemia reperfusion mouse brain injury protective effect It prepares Kunming mouse MCAO ischemia-reperfusion models and causes cerebral ischemia and Brain edema, give glibenclamide and sweet wormwood amber Ester injection treatment, does positive control, after the treatment by infarction of brain volume ratio, edema volume with Edaravone Injection Than, the evaluation of measuring Sur1 inhibitor glibenclamide and sweet wormwood of Neuroscore and brain tissue inflammation's factor TNF-α and IL-6 Plain Sesquiterpene lactones compound is combined the therapeutic effect to cerebral apoplexy. 1, experimental animal and grouping Kunming mouse 90 (is purchased from Kunming Medical University's SPF Animal Lab), male, 25-28g.It is randomly divided into 9 Experimental group, every group 10, including： 1. sham-operation group：Arteria carotis communis, external carotid artery and internal carotid, but the not embolism cerebrovascular are isolated, iv approach is given Give the solvent (0.9% physiological saline) for the treatment of group's equivalent； 2. model control group：It prepares cerebral ischemia (MCAO) and fills model again, intravenous injection (iv) approach is given with treatment group etc. The solvent (0.9% physiological saline) of amount； 3. 10 μ g/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide； 4. 30 μ g/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide； 5. Artesunate 25mg/kg groups：Cerebral ischemic model is prepared, iv approach gives Artesunate； 6. Artesunate 50mg/kg groups：Cerebral ischemic model is prepared, iv approach gives Artesunate； 7. 10 μ g/kg+ Artesunate 25mg/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide And Artesunate 8. 30 μ g/kg+ Artesunate 50mg/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide And Artesunate Positive drug 9. (Edaravone 3mg/kg) control group：Cerebral ischemic model is prepared, iv approach gives Edaravone； 2, prepared by model： Arterial occlusion (MCAO) model in transience mouse brain is prepared using line brush.Its operating procedure is as follows：First will Record of weighing after healthy Kunming mouse number is lain on the back and is fixed on operating table with 4% chloraldurate intraperitoneal injection of anesthesia, 60 DEG C of incandescent lamp irradiations are being placed at operating table 30cm, and mouse temperature is made to maintain 37 DEG C or so.It cuts neck and hits exactly skin Skin detaches subcutaneous tissue and is bubbled body of gland, exposes ectotrachea muscle, and separating mouse right muscles, to isolate right side neck total Artery (CCA), external carotid artery (ECA) and internal carotid (ICA), in CCA proximal parts and ECA distal ends, (distance CCA crotches are about 5mm) place's hanging wire is spare.Then distal end ligatures CCA and ECA.With No. 2 syringe needles from CCA away from slotting from the about 0.5cm of the forks ECA and ICA Enter line bolt.Insertion depth touches bolt line to when about 1cm (at mark point) with ophthalmic tweezers, feels that the when of having resistance indicates to have been inserted into Position, at this moment gently fastens bolt line, skin suture with filament.Nylon embolus line is extracted after 2h, so that its blood flow is led to again, is divided cage to put mouse Raising, pays attention to warming in steaming again. 3, it is administered Glibenclamide is administered successively in three times for 0h after ischemia-reperfusion (being administered immediately after pulling out Outlet bolt), 3h and 6h, Administering mode is tail vein injection (iv).Artesunate is 0h Bolos intravenous administrations after ischemia-reperfusion.Artesunate is with 5% NaHCO3Dissolving, glibenclamide are dissolved by heating with 1% meglumine. 4, experimental endpoints sample collection It before putting to death rat, detaches abdominal aorta and takes blood 4mL, after standing 1h, 3000r/min centrifuges 10min, draws blood Clearly.- 20 DEG C of refrigerators are put into, TNF-α and IL-6 concentration are measured using Double antibody sandwich-ELISA (ELISA).Strictly It is operated by ELISA kit operation instructions. 5, evaluation index 5.1 Neuroscore：After rat operation for 24 hours, neurological deficit score is carried out to experimental animal and recorded, methods of marking For Longa methods, standards of grading are as follows： 0 point：Impassivity defective symptom； 1 point：Offside forelimb cannot stretch completely； 2 points：It turn-takes to offside； 3 points：It walks and is tilted to offside； 4 points：It spontaneous cannot walk, the loss of consciousness. 5.2 cerebral infarction volumes detect 5.3 brain water contents measure：Swelling volume and swelling percentage are calculated using swelling volumetric method. 6, statistical analysis Each level area of brain section, infarct size etc. are measured using Imagepro plus6.0.Pass through Graphpad 5 softwares of Prism are for statistical analysis to data, one-way or two-way repeat measure ANOVA post-hoc Dunnett ' test carry out comparison among groups, and the standard of significant difference is 0.05. 7, experimental result 7.1 cerebral infarction volumes detect：By ischemia-reperfusion for 24 hours after mouse row excessively anaesthetize, directly take brain after broken end, set In in mouse brain mold, then it is sliced from front to back by brain is coronal, is divided into 4, is put in 1.0%TTC solution, 37 DEG C are protected from light 10min dyeing is incubated, infarcted region is not colored, and normal cerebral tissue dyes red.Then it takes pictures, passes through to every a piece of brain piece Imagepro plus6.0 image analysis systems measure each level area, ischemic areas, half brain area of ischemic side (homonymy area), Half brain area (to lateral area) of non-ischemic side calculates respective volume (mm in conjunction with slice thickness3), and calculate Infarction volume and account for offside The percentage of cerebral hemisphere volume.The results are shown in Figure 1, model group, positive controls (Edaravone -3mg/kg), Ge Lieben Urea 10ug/kg, glibenclamide 30ug/kg, Artesunate 25mg/kg groups, Artesunate 50mg/kg groups, glibenclamide 10ug/kg + Artesunate 25mg/kg combinations group, glibenclamide 30ug/kg+ Artesunate 50mg/kg combination groups are compared with sham-operation group, all There is different degrees of infarct, shows that MCAO ischemia-reperfusion models are successfully established；Wherein, compared with model group, Artesunate 25mg/kg groups, Artesunate 50mg/kg groups, glibenclamide 10ug/kg+ Artesunate 25mg/kg combination group glibenclamides 30ug/kg+ Artesunate 50mg/kg combinations groups can be substantially reduced infarct size, and be better than positive controls. 7.2 experiments calculate swelling volume and swelling percentage using swelling volumetric method.As shown in Fig. 2, compared with model group, Glibenclamide 30ug/kg and glibenclamide 30ug/kg+ Artesunate 50mg/kg combinations groups can significantly mitigate Brain edema, and be better than Positive controls. 7.3 Neurological deficits Neurological deficits reflect the case where drug improves prognosis to mouse cerebral apoplexy, and the results are shown in Table 1： Table 1, embodiment MACO mouse Nerves neurological deficit score (n=6) Group Neurological deficits (average value) Sham-operation group 0 Model group 3.2 Glibenclamide 10ug/kg 2.1 Glibenclamide 30ug/kg 2.0 Artesunate 25mg/kg 2.4 Artesunate 50mg/kg 2.1 Glibenclamide 10ug/kg+ Artesunates 25mg/kg 1.6 Glibenclamide 30ug/kg+ Artesunates 50mg/kg 1.2 Edaravone 3mg/kg 1.8 TNF-α and IL-6 are horizontal in 7.4 serum Using enzyme-linked immunization, detects separate groups of mice serum cytokines TNF-α and IL-6 is horizontal.As a result it shows：With Normal group mouse is compared, model group TNF-α and IL-6 contents significantly increase (###p<0.001)；Compared with model group mouse, Artesunate group, glibenclamide 10ug/kg+ Artesunates 25mg/kg combinations group and glibenclamide 30ug/kg+ Artesunates 50mg/kg combinations group can significantly reduce TNF-α and IL-6 contents in serum (***p<0.001,*p<0.05).As a result see Fig. 3： 8, conclusion Show from the mouse MCAO ischemia-reperfusion models treatment drug effect evaluation result carried out in animal level green in the present invention Artemisic succinate, glibenclamide and Artesunate combination can significantly reduce Serum TNF-α and IL-6 contents, reduce brain edema and infraction, Especially combination group can improve prognosis, improve life in patients, have good development and application values. Above-mentioned experiment is also carried out to the other combination products of the present invention, the result obtained is similar to above-mentioned test result. The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, the technology people of any familiar present invention Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little variation or be modified to The equivalent embodiment of equivalent variations, it is right according to the technical essence of the invention as long as being the content without departing from technical solution of the present invention Any simple modification, equivalent change and modification made by above example, in the range of still falling within the present invention program. Claims (9) 1. a kind of for preventing and/or treating the combination product of cerebral ischemia, which is characterized in that the combination product be containing The pharmaceutical composition of Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound； Or the group for the first medicament containing Sur1 inhibitor and the second medicament containing qinghaosu Sesquiterpene lactones compound It closes. 2. combination product according to claim 1, which is characterized in that Sur1 inhibitor and blueness in the pharmaceutical composition The mass ratio of artemisin Sesquiterpene lactones compound is (1~3):(2500~5000)； In the combination in the first medicament in Sur1 inhibitor and second medicament qinghaosu Sesquiterpene lactones compound matter Amount is than being (1~3):(2500~5000). 3. combination product according to claim 1 or 2, which is characterized in that the pharmaceutical composition be Sur1 inhibitor, Pharmaceutically acceptable dosage form is made with pharmaceutically acceptable auxiliary material in qinghaosu Sesquiterpene lactones compound, preferably injects Agent, more preferable injection or freeze-dried powder. 4. combination product according to claim 1 or 2, which is characterized in that first medicament be Sur1 inhibitor with Pharmaceutically acceptable dosage form is made in pharmaceutically acceptable auxiliary material； The second medicament is that qinghaosu Sesquiterpene lactones compound and pharmaceutically acceptable auxiliary material are made and pharmaceutically may be used The dosage form of receiving, optimizing injection, more preferable injection or freeze-dried powder. 5. combination product according to any one of claims 1-4, which is characterized in that the Sur1 inhibitor is selected from lattice Arrange one or more of this urea, orinase, Repaglinide, meglitinide, midaglizole or Glimepiride. 6. according to the combination product described in claim 1-5 any one, which is characterized in that the qinghaosu sesquiterpene lactone Class compound is the sesquiterpene lactone extracted from artemisia annua and the derivative of qinghaosu. 7. combination product according to claim 6, which is characterized in that the qinghaosu Sesquiterpene lactones compound is Artesunate, dihydroartemisinine or Artemether. 8. the combination product described in claim 1-7 any one is preparing the application in treating Imaging in Patients with Cerebral Ischemia Disease drug. 9. application according to claim 8, which is characterized in that the cerebral ischemia is cerebral apoplexy. 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Priority And Related Applications Priority Applications (1) ApplicationPriority dateFiling dateTitle CN201810246790.XA2018-03-232018-03-23A kind of combination product and its application for preventing and/or treating cerebral ischemia Applications Claiming Priority (1) ApplicationFiling dateTitle CN201810246790.XA2018-03-23A kind of combination product and its application for preventing and/or treating cerebral ischemia https://patents.google.com/patent/CN108310387A/en 一种用于预防和/或治疗脑缺血的组合产品及其应用 技术领域 本发明属于生物医药技术领域，具体地说，涉及一种用于预防和/或治疗脑缺血的组合产品及其应用。 背景技术 缺血性脑卒中具有高发病率、高死亡率、高致残率、高复发率、高医疗花费几个特点，是危害我国居民健康的重大慢性病。临床数据统计显示，全球脑卒中死亡率已经超过癌症成为单病种死亡率最高的疾病。众所周知，炎症反应是脑缺血病理损伤过程中的重要组成部分，也是加重脑缺血损伤的潜在因素。除此之外，缺血性脑卒中常伴有脑水肿和出血性转化，其中脑水肿是缺血性脑卒中最严重的并发症，卒中患者死亡中80％死于卒中后期的恶性脑水肿。因此炎症及脑水肿的防治对缺血性脑卒中的治疗有重要意义。 磺脲类受体1(sulfonylurea receptor 1，Sur1)-瞬时受体电位通道M4型(transient receptor potential melastatin 4，Trpm4)通道对于脑水肿的形成起着重要作用。相关文献报道，缺血性脑卒中发生4-8小时是脑水肿形成的关键时期，在这时期缺血部位脑血管内壁细胞膜Sur1-Trpm4通道的Sur1受体会出现应激性高表达，大量表达的Sur1受体导致Sur1-Trpm4通道激活。Sur1-Trpm4通道的激活增加阳离子特别是钠离子进入细胞内，由于渗透压的缘故驱动水大量涌入细胞内，引起缺血部位细胞乃至组织发生水肿，是卒中患者出现脑水肿的关键病理机制。Sur1抑制剂与Sur1受体结合抑制这个通道的开放，从而有效地抑制脑水肿的形成。代表性Sur1抑制剂格列本脲针对卒中后脑水肿的治疗已在美国进入临床III期。 青蒿素及其衍生物是我国科学工作者研究开发的一类抗疟新药，目前广泛应用于疟疾的治疗。随着研究的不断深入，除了其抗疟作用外，科学家还相继发现了青蒿素及其衍生物在其他方面的药理作用，包括抗肿瘤作用、对免疫细胞的作用、抗纤维化作用、抗肥胖、抗糖尿病以及抗真菌作用等，其中抗疟作用和抗肿瘤作用最为引人关注。青蒿素及其衍生物在炎性方面的研究主要基于其对自身免疫性疾病的治疗作用，如红斑狼疮和类风湿关节炎，研究显示其对多个炎性相关靶蛋白包括NF-Kb和细胞因子，例如IL-1、IL-6和IL-8等均有调控作用。但青蒿素及其衍生物在治疗脑缺血性疾病方面的应用至今未见研究报道。 有鉴于此特提出本发明。 发明内容 本发明要解决的技术问题在于克服现有技术的不足，提供一种用于预防和/或治疗脑缺血的组合产品及其应用，该组合产品的成分之间具备协同增效的作用。 为解决上述技术问题，本发明采用如下技术方案： 一种用于预防和/或治疗脑缺血的组合产品，其中，所述的组合产品为含有Sur1抑制剂和青蒿素倍半萜内酯类化合物的药物组合物； 或为含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂的组合。 具体地说，一方面，本发明所提供的用于预防和/或治疗脑缺血的组合产品，可以是将Sur1抑制剂和青蒿素倍半萜内酯类化合物放在一起形成一种药物组合物，该药物组合物可进一步与药学上可接受的辅料制成一种复方制剂。 另一方面，本发明所提供的用于预防和/或治疗脑缺血的组合产品，也可以是将含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂进行联合用药。在联合用药时，第一药剂与第二药剂的用药顺序不分先后，可以先用第一药剂，也可以先用第二药剂，还可以两个药剂同时使用。 进一步的，所述的药物组合物中Sur1抑制剂和青蒿素倍半萜内酯类化合物的质量比为(1～3):(2500～5000)； 所述的组合中第一药剂中Sur1抑制剂和第二药剂中青蒿素倍半萜内酯类化合物的质量比为(1～3):(2500～5000)。 进一步的，所述的药物组合物为Sur1抑制剂、青蒿素倍半萜内酯类化合物与药学上可接受的辅料制成药学上可接受的剂型，优选注射剂，更优选注射液或冻干粉针。 其辅料种类和制备方法可参照现有技术。 进一步的，所述的第一药剂为Sur1抑制剂与药学上可接受的辅料制成药学上可接受的剂型； 所述的第二药剂为青蒿素倍半萜内酯类化合物与药学上可接受的辅料制成药学上可接受的剂型，优选注射剂，更优选注射液或冻干粉针。 本发明中，可以将含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂进行联合用药。所述的第一药剂或第二药剂可以是现有技术中已有的制剂，也可以是将Sur1抑制剂或青蒿素倍半萜内酯类化合物分别添加药学上可接受的辅料分别制成药学上可接受的剂型。辅料种类和各剂型的制备方法可参照现有技术。 进一步的，所述的Sur1抑制剂选自格列本脲、甲苯磺丁脲、瑞格列奈、美格列奈、咪格列唑或格列美脲中的一种或几种。 进一步的，所述的青蒿素倍半萜内酯类化合物是从黄花蒿中提取的倍半萜内酯及青蒿素的衍生物。 进一步的，所述的青蒿素倍半萜内酯类化合物为青蒿琥酯、双氢青蒿素或蒿甲醚。 本发明还提供所述的组合产品在制备治疗脑缺血疾病药物中的应用。 所述的脑缺血为脑卒中。 采用上述技术方案后，本发明与现有技术相比具有以下有益效果： 本发明提供一种含有Sur1抑制剂和青蒿素倍半萜内酯类化合物，其中Sur1抑制剂可通过抑制sur1减轻脑水肿，而青蒿素倍半萜内酯类化合物可通过抑制炎症因子及相关蛋白发挥抗炎作用。两者以适当比例配合使用时具有协同增效的作用。将Sur1抑制剂和青蒿素倍半萜内酯类化合物以适当比例联合用药，可以比单用Sur1抑制剂或者青蒿素倍半萜内酯类化合物对脑缺血性疾病有更好的治疗效果。 下面结合附图对本发明的具体实施方式作进一步详细的描述。 附图说明 图1示脑梗死体积百分比，假手术组vs模型组，###p＜0.001；给药组vs模型组，**p＜0.01，*p＜0.05； 图2示脑水肿体积百分比，假手术组vs模型组，###p<0.001；给药组vs模型组，**p＜0.01； 图3示血清TNF-α和IL-6表达水平，假手术组vs模型组，###p＜0.001；给药组vs模型组，***p＜0.001，*p＜0.05； 需要说明的是，这些附图和文字描述并不旨在以任何方式限制本发明的构思范围，而是通过参考特定实施例为本领域技术人员说明本发明的概念。 具体实施方式 为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述，以下实施例用于说明本发明，但不用来限制本发明的范围。 以下为实施例1至实施例7，各实施例中药物组合物的组成见表1所示，其制备方法为按所述质量称取各物质，然后将其混合均匀，即得药物组合物。 表1 上述药物组合物可进一步添加药学上可接受的辅料制备成药学上可接受的剂型，优选注射剂，包括但不限于注射液或冻干粉针。 试验例1、格列本脲与青蒿琥酯对脑缺血再灌小鼠脑损伤保护作用的研究 制备昆明种小鼠MCAO缺血再灌模型造成脑缺血及脑肿胀，给予格列本脲及青蒿琥酯注射治疗，用依达拉奉注射液做阳性对照，在治疗后通过对大脑梗死体积比、水肿体积比、神经功能评分、及脑组织炎症因子TNF-α和IL-6的测定评价Sur1抑制剂格列本脲及青蒿素倍半萜内酯类化合物联用对脑卒中的治疗作用。 1、实验动物及分组 昆明种小鼠90只(购自昆明医科大学SPF动物实验室)，雄性，25-28g。随机分为9个实验组，每组10只，包括： ①假手术组：分离出颈总动脉、颈外动脉及颈内动脉，但不栓塞脑血管，iv途径给予治疗组等量的溶剂(0.9％生理盐水)； ②模型对照组：制备脑缺血(MCAO)再灌模型，静脉注射(iv)途径给予与治疗组等量的溶剂(0.9％生理盐水)； ③格列本脲10μg/kg组：制备脑缺血模型，iv途径给予格列本脲； ④格列本脲30μg/kg组：制备脑缺血模型，iv途径给予格列本脲； ⑤青蒿琥酯25mg/kg组：制备脑缺血模型，iv途径给予青蒿琥酯； ⑥青蒿琥酯50mg/kg组：制备脑缺血模型，iv途径给予青蒿琥酯； ⑦格列本脲10μg/kg+青蒿琥酯25mg/kg组：制备脑缺血模型，iv途径给予格列本脲及青蒿琥酯 ⑧格列本脲30μg/kg+青蒿琥酯50mg/kg组：制备脑缺血模型，iv途径给予格列本脲及青蒿琥酯 ⑨阳性药(依达拉奉3mg/kg)对照组：制备脑缺血模型，iv途径给予依达拉奉； 2、模型制备： 采用线栓法制备短暂性小鼠大脑中动脉闭塞(MCAO)模型。其操作步骤如下：先将健康昆明种小鼠编号后称重记录，以4％的水合氯醛腹腔注射麻醉，仰卧固定于手术台上，在距离手术台30cm处放置60℃白炽灯照射，使小鼠体温维持在37℃左右。切开颈部正中皮肤，分离皮下组织及鼓泡腺体，暴露出气管外层肌肉，分离小鼠右侧肌肉、分离出右侧颈总动脉(CCA)、颈外动脉(ECA)和颈内动脉(ICA)，在CCA近心端及ECA远心端(距离CCA分叉处约5mm)处挂线备用。然后远心端结扎CCA和ECA。用2号针头从CCA距ECA和ICA岔口约0.5cm处插入线栓。插入深度到约1cm(标记点处)时用眼科镊轻推栓线，感觉到有阻力时即表示已插到位，这时用细线轻轻系牢栓线，缝合皮肤。2h后拔出尼龙栓线，使其血流再通，将小鼠分笼放回笼内饲养，注意保暖。 3、给药 格列本脲为缺血再灌注后0h(即拔出线栓后立即给药)、3h和6h分三次依次给药，给药方式为尾静脉注射(iv)。青蒿琥酯为缺血再灌注后0h静注给药。青蒿琥酯用5％的NaHCO3溶解，格列本脲均用1％的葡甲胺加热溶解。 4、实验终点样品采集 在处死大鼠前，分离腹主动脉并取血4mL，静置1h后，3000r/min离心10min，吸取血清。放入-20℃冰箱，采用双抗体夹心酶联免疫吸附法(ELISA)测定TNF-α和IL-6浓度。严格按ELISA试剂盒使用说明书操作。 5、评价指标 5.1神经功能评分：大鼠手术后24h，对实验动物进行行为学评分并记录，评分方法为Longa法，评分标准如下： 0分：无神经缺损症状； 1分：对侧前肢不能完全伸直； 2分：向对侧转圈； 3分：行走向对侧倾斜； 4分：不能自发行走，意识丧失。 5.2脑梗死体积检测 5.3脑含水量测定：采用肿胀体积法计算肿胀体积及肿胀百分比。 6、统计学分析 采用Imagepro plus6.0测定脑切片各层面面积、梗死面积等。通过GraphpadPrism 5软件对数据进行统计分析，one-way或two-way repeat measure ANOVA post-hocDunnett’test进行组间比较，显著性差异的标准为0.05。 7、实验结果 7.1脑梗死体积检测：将缺血再灌注24h后的小鼠行过量麻醉，直接断头后取脑，置于小鼠脑模具内，然后将大脑冠状从前向后切片，分为4片，放于1.0％TTC溶液中，37℃避光温育10min染色，梗死区不着色，正常脑组织染成红色。然后对每一片脑片进行拍照，经Imagepro plus6.0图像分析系统测量各层面面积、缺血面积、缺血侧半脑面积(同侧面积)、未缺血侧半脑面积(对侧面积)，结合切片厚度计算相应体积(mm3),并计算梗死体积占对侧大脑半球体积的百分比。结果如图1所示，模型组、阳性对照组(依达拉奉-3mg/kg)、格列本脲10ug/kg、格列本脲30ug/kg、青蒿琥酯25mg/kg组、青蒿琥酯50mg/kg组、格列本脲10ug/kg+青蒿琥酯25mg/kg联用组、格列本脲30ug/kg+青蒿琥酯50mg/kg联用组与假手术组相比，都出现了不同程度梗死，表明MCAO缺血再灌模型成功建立；其中，与模型组相比，青蒿琥酯25mg/kg组、青蒿琥酯50mg/kg组、格列本脲10ug/kg+青蒿琥酯25mg/kg联用组格列本脲30ug/kg+青蒿琥酯50mg/kg联用组能显著减小梗死面积，且优于阳性对照组。 7.2实验采用肿胀体积法计算肿胀体积及肿胀百分比。如图2所示，与模型组比较，格列本脲30ug/kg和格列本脲30ug/kg+青蒿琥酯50mg/kg联用组能显著减轻脑肿胀，且优于阳性对照组。 7.3神经行为学评分 神经行为学评分反映药物对小鼠脑卒中改善预后的情况，结果见表1所示： 表1、实施例MACO小鼠神经行为学评分(n＝6) 组别 神经行为学评分(平均值) 假手术组 0 模型组 3.2 格列苯脲10ug/kg 2.1 格列苯脲30ug/kg 2.0 青蒿琥酯25mg/kg 2.4 青蒿琥酯50mg/kg 2.1 格列苯脲10ug/kg+青蒿琥酯25mg/kg 1.6 格列苯脲30ug/kg+青蒿琥酯50mg/kg 1.2 依达拉奉3mg/kg 1.8 7.4血清中TNF-α和IL-6水平 采用酶联免疫法，检测不同组小鼠血清细胞因子TNF-α和IL-6水平。结果显示：与正常对照组小鼠比较，模型组TNF-α和IL-6含量显著增高(###p<0.001)；与模型组小鼠比较，青蒿琥酯组、格列本脲10ug/kg+青蒿琥酯25mg/kg联用组及格列本脲30ug/kg+青蒿琥酯50mg/kg联用组可显著降低血清中TNF-α和IL-6含量(***p<0.001，*p<0.05)。结果见图3： 8、结论 从在动物水平进行的小鼠MCAO缺血再灌模型治疗药效评价结果表明，本发明中青蒿琥酯、格列本脲与青蒿琥酯联用能显著降低血清TNF-α和IL-6含量，减少脑水肿及梗塞，特别是联用组能改善预后、提高患者生存质量，具有很好的开发应用价值。 对本发明其它组合产品也进行了上述试验，其获得的结果与上述试验结果相似。 以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本发明的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许变动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。 Claims (9) 1.一种用于预防和/或治疗脑缺血的组合产品，其特征在于，所述的组合产品为含有Sur1抑制剂和青蒿素倍半萜内酯类化合物的药物组合物； 或为含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂的组合。 2.根据权利要求1所述的组合产品，其特征在于，所述的药物组合物中Sur1抑制剂和青蒿素倍半萜内酯类化合物的质量比为(1～3):(2500～5000)； 所述的组合中第一药剂中Sur1抑制剂和第二药剂中青蒿素倍半萜内酯类化合物的质量比为(1～3):(2500～5000)。 3.根据权利要求1或2所述的组合产品，其特征在于，所述的药物组合物为Sur1抑制剂、青蒿素倍半萜内酯类化合物与药学上可接受的辅料制成药学上可接受的剂型，优选注射剂，更优选注射液或冻干粉针。 4.根据权利要求1或2所述的组合产品，其特征在于，所述的第一药剂为Sur1抑制剂与药学上可接受的辅料制成药学上可接受的剂型； 所述的第二药剂为青蒿素倍半萜内酯类化合物与药学上可接受的辅料制成药学上可接受的剂型，优选注射剂，更优选注射液或冻干粉针。 5.根据权利要求1-4任意一项所述的组合产品，其特征在于，所述的Sur1抑制剂选自格列本脲、甲苯磺丁脲、瑞格列奈、美格列奈、咪格列唑或格列美脲中的一种或几种。 6.根据权利要求1-5任意一项所述的组合产品，其特征在于，所述的青蒿素倍半萜内酯类化合物是从黄花蒿中提取的倍半萜内酯及青蒿素的衍生物。 7.根据权利要求6所述的组合产品，其特征在于，所述的青蒿素倍半萜内酯类化合物为青蒿琥酯、双氢青蒿素或蒿甲醚。 8.权利要求1-7任意一项所述的组合产品在制备治疗脑缺血疾病药物中的应用。 9.根据权利要求8所述的应用，其特征在于，所述的脑缺血为脑卒中。 Patent Citations (2) Publication numberPriority datePublication dateAssigneeTitle CN101043891A *2004-09-182007-09-26巴尔的摩马里兰大学靶向NCCa－ATP通道的治疗剂及其使用方法 US20080161324A1 *2006-09-142008-07-03Johansen Lisa MCompositions and methods for treatment of viral diseases Family To Family Citations * Cited by examiner, † Cited by third party Non-Patent Citations (2) Title LU ET AL.: ""Artesunate suppresses oxidative and inflammatory processes by activating Nrf2 and ROS‐dependent p38 MAPK and protects against cerebral ischemia‐reperfusion injury"", 《MOLECULAR MEDICINE REPORTS》 * 吴舟: ""格列本脲延长低温对大鼠脑局灶缺血再灌注损伤的治疗时间窗"", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * * Cited by examiner, † Cited by 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The invention provides a novel dihydroartemisinin derivative, which can be used as an active ingredient to prepare a medicine for preventing and treating ischemic cerebral apoplexy, and the medicine can pass through a blood brain barrier more easily and has higher brain tissue medicine concentration. 本发明提供了一种新的双氢青蒿素衍生物，以该双氢青蒿素衍生物为活性成分可制备成预防、治疗缺血性脑卒中的药物，而且所述药物更容易通过血脑屏障，具有更高的脑组织药物浓度。 Dihydroartemisinin derivative, medicine and application thereof in preparation of medicine for preventing and treating cerebrovascular diseases Technical Field The invention relates to a dihydroartemisinin derivative and a pharmaceutical application thereof. Background With the aging process of China, cerebrovascular diseases become the first cause of death disability. The main cause is that the nerve cells die from ischemia and hypoxia due to the interruption of blood flow caused by cerebral vascular embolism or thrombosis, and the severity of the illness is closely related to the infarct volume of brain tissues. Neural stem cells (Neural Stem Cells, NSCs), which are mainly present in the subtubular region of the lateral ventricle wall and the dentate gyrus region of the hippocampus, are a group of special cells that can self-renew, stem maintain and differentiate into functional neurons. Since neurons are terminally differentiated cells and cannot self-renew, the neurons can only repair functions through proliferation and differentiation of neural stem cells after ischemic death, and how to activate rapid proliferation of endogenous neural stem cells to repair infarcted brain tissues in the research of cerebrovascular diseases is always a hot spot field. The subject group is directed towards artemisinin and its use in the treatment of neurological related disorders. CN104523679a provides the use of dihydroartemisinin in the treatment of cerebrovascular diseases. Although dihydroartemisinin is found to have a promoting effect on neural stem cell proliferation, neural stem cell proliferation can be induced in vitro, and ischemic sites of cerebral infarction can be reduced in vivo. However, studies have shown that long-term low doses of dihydroartemisinin may lead to side effects such as bone marrow suppression, peripheral red blood cell depletion and extramedullary hematopoiesis of the spleen, while reduced doses may effectively avoid the occurrence of related side effects [ Yin Jiye. Toxicology reevaluation of two artemisinin derivatives and its cardiotoxicity mechanism study [ D ]. National discharge military medical sciences, 2014 ]. In addition, as the research is in progress, dihydroartemisinin belongs to a water-soluble drug, and can pass through the blood brain barrier, but has limited transmittance, and higher blood drug concentration is required to reach the brain tissue concentration required by neuroprotection [ Yepu He et al, synergistic integration of dihydro-artemsinin with gamma-aminobutyric acid results in a more potential anti-depressant. Bioorg chem.2021.110:104769 ]. In conclusion, the search of the dihydroartemisinin derivative can achieve higher blood brain barrier permeability, and has important clinical significance in reducing side reactions generated in the treatment of cerebrovascular diseases. Disclosure of Invention Based on the problem, through intensive research, the invention aims to provide a novel dihydroartemisinin derivative which has the function of treating and preventing cerebrovascular diseases, improves the problem of low permeability of dihydroartemisinin blood brain barrier and improves the drug concentration in brain tissues. The aim of the invention is achieved by the following measures: a dihydroartemisinin derivative, which has the structural formula: the invention also provides a medicine. A medicament comprising a dihydroartemisinin derivative as described above. The medicament may also comprise one or more pharmacologically acceptable auxiliary materials including diluents, excipients, fillers, wetting agents, absorption enhancers, surfactants, lubricants or stabilizers and the like which are conventional in the pharmaceutical arts. The medicine is preferably prepared into aqueous solution or powder and other medicine forms suitable for injection; more preferably an aqueous solution, the drug concentration is 150mg/kg. The medicine can be used by intravenous drip or intramuscular injection, and is generally applied for 1 time in 1 day, 40mg each time, and 14 days are a course of treatment. The dosage is reduced by 30% compared with the conventional dosage of dihydroartemisinin (60 mg). It is another object of the present invention to provide the use of the above dihydroartemisinin derivatives. The application of the dihydroartemisinin derivative in the medicaments for treating and preventing the cerebrovascular diseases. Furthermore, the dihydroartemisinin derivative is applied to the preparation of medicines for treating and preventing ischemic cerebral apoplexy. Furthermore, the dihydroartemisinin derivative is applied to the preparation of medicines for relieving or eliminating cerebral infarction and ischemia. The application of the dihydroartemisinin derivative in preparing medicines for promoting proliferation of neural stem cells. Further, the use of said dihydroartemisinin derivatives to promote proliferation of brain subventricular zone (Subventricular Zone, SVZ) neural stem cells (Neural Stem Cells, NSCs). The dihydroartemisinin derivative has a promoting effect on the proliferation of the neural stem cells; in vitro, neural stem cell proliferation can be induced; can effectively reduce ischemic focus of cerebral infarction and improve neurological deficit caused by cerebral apoplexy. The dihydroartemisinin derivative can be used as an active ingredient to prepare a medicine for treating ischemic cerebral apoplexy, and the medicine can pass through the blood brain barrier more easily, so that the medicine has higher brain tissue medicine concentration. Advantageous effects 1. After cerebral ischemia, a large number of functional nerve cells die in a short period of time due to the special tolerance of neurons to blood oxygen, which has an extremely adverse effect on prognosis. The dihydroartemisinin derivative provided by the invention maintains the related functions of dihydroartemisinin in treating the related diseases, and particularly can promote endogenous NSCs to activate, proliferate and differentiate into nerve precursor cells (neurobalasts) and migrate to lesion sites after the nerve cells in an ischemic area are damaged, and the dihydroartemisinin derivative is re-integrated in a damaged nerve network system, restores a damaged nerve network structure and can replace necrotic neurons to play corresponding nerve functions. The dihydroartemisinin derivative functions by early activation (figure 2) of proliferation of NSCs endogenous to neural stem cells instead of periischemic necrotic neurons, and can be used for treating cerebral infarction. 2. The dihydroartemisinin derivative provided by the invention not only has the efficacy, but also has better blood brain barrier transmittance (figure 4) than dihydroartemisinin, can have higher drug concentration for central nervous system diseases, can effectively reduce the drug dosage, improve the drug curative effect, and further reduce the toxic and side effects. Drawings FIG. 1 chemical structural Change in the preparation of Dihydroartemisinin derivatives (Dihydroartemisinin derivatives, DD) in example 1 FIG. 2 example 2 immunofluorescence results show: the NSC proliferation number (Nestin/Ki 67 double-dyeing) of the DD group is obviously increased compared with that of the control group; FIG. 3 MRI photograph of the MCAO animal of example 3 after 72 hours of surgery, it can be seen that the cerebral infarction focus of the DD group animal is significantly reduced compared with the control group and the Dihydroartemsinin (DHA) group FIG. 4 high performance liquid chromatography of MCAO animals of example 4 after 72h of surgery to determine plasma and brain tissue drug concentrations; A. determining a chromatogram of DHA in plasma; B. DD determination chromatogram in brain tissue; C. no significant difference in plasma concentration versus the two groups; D. the concentration in brain tissue is significantly higher than in DHA. Detailed Description The invention will be described in further detail with reference to the drawings and examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control. Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not noted, according to conventional conditions or conditions suggested by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's knowledge. EXAMPLE 1 preparation of Dihydroartemisinin derivative (Dihydroartemisinin derivatives, DD) 1.10 preparation of beta- (2-bromoethoxy) dihydroartemisinin 3.103g (24 mmol) of 2-bromoethyl alcohol and 100mL of tetrahydrofuran are introduced into a 250mL round bottom flask, followed by 4mL of BF3.Et2O (C) under ice-bath conditions 4 H 10 BF 3 O, boron trifluoride etherate, commercially available) and 5.690g (20 mmol) of dihydroartemisinin (C) 15 H 24 O 5 ). The mixture was stirred in an ice bath for 1.5 hours. The reaction progress was monitored by TLC. The reaction is completed, saturated NaHCO is added respectively 3 Solution and 50mL EtOAc (CH) 3 COOC 2 H 5 ) After extraction of the layers, the aqueous layer was extracted with EtOAc (30 ml×2) and the organic layers were combined. The organic layer was washed with 40mL of saturated brine solution, anhydrous solid Na 2 SO 4 After drying and filtration, the organic solvent was distilled under reduced pressure. The crude product obtained was recrystallized (petroleum ether was added dropwise to EtOAc dissolved in sample), left to stand, filtered, and dried in vacuo to give white crystals. 2. Preparation of dihydroartemisinin derivatives 10 beta- (2-bromoethoxy) dihydroartemisinin, CH 3 CN，KI，K 2 CO 3 And furbenamine% Sigma-Aldrich company) was added to a 100mL round bottom flask, and the mixture was reacted by heating at a controlled temperature (50 ℃). The reaction progress was monitored by TLC. Then 15mL of methylene chloride and 20mL of saturated NaCl solution were added. After the separation by extraction, the aqueous layer was extracted with dichloromethane (10 ml×2) and the organic layers were combined. The organic layer was washed with 20mL of saturated brine solution, anhydrous solid Na 2 SO 4 Drying; after filtration, dichloromethane was distilled under reduced pressure. After silica gel column chromatography (petroleum ether mixed EtOAc) the pure product was obtained. The structure change process of the compound is shown in figure 1. Respectively obtaining under the conditions of 600MHz and 151MHz of an Agilent nuclear magnetic resonance apparatus 1 H spectrum 13 C spectrum, the compound is measured by high resolution mass spectrum using a Waters SYNPAT G2 instrument, and the spectrum information is as follows: 1H NMR (600 MHz, DMSO) d 7.75 (s, 1H), 6.57 (s, 1H), 6.52 (s, 1H), 5.40 (s, 1H), 4.71 (s, 1H), 4.20 (s, 2H), 3.89 (s, 1H), 3.55 (s, 1H), 3.08 (s, 2H), 2.42 (s, 1H), 2.18 (s, 1H), 2.01 (s, 1H), 1.80 (s, 1H), 1.66 (M, 2H), 1.54 (s, 1H), 1.32 (M, 6H), 1.14 (s, 1H), 0.88 (M, 7H) 13C NMR (MHz, DMSO) d 146.84,143.96,111.41,110.87,103.43,101.06,86.89,80.34,63.65,51.90,45.75,43.83,42.83,36.46,36.01,34.09,30.27,25.62,24.22,23.95,20.11,12.62.HRMS M/z [ M+H 151]+:408.2375(Calcd for C22H33NO6:407.230788)。 EXAMPLE 2 Effect of Dihydroartemisinin Derivative (DD) on proliferation of neural Stem cells under ex vivo conditions Pregnant 14.5d SD rats were selected, 5% chloral hydrate was anesthetized intraperitoneally, the peritoneum was opened, embryos were removed, scalp, skull and dura were removed, and the rat brain was stripped. Under a dissecting microscope, longitudinal cutting is carried out by taking the olfactory bulb as a central sagittal position, and materials are obtained in the subventricular zone. Proliferation medium (DMEM/F12, bFGF 20ng/mL, EGF 20ng/mL,2% B27) was added, and after filtration through a 200 mesh screen, trypan blue staining was performed, and the cell count was adjusted to 1X 10 6 Transfer to a culture flask, put into a 5% CO2 incubator at a constant temperature of 37 ℃ for culture, and passaged once in three days. Taking NSCs grown in logarithmic phase, digesting with 0.25% EDTA trypsin for 2min, adjusting cell number to 8000/100 μl after stopping digestion with 10% DMEM/F12, inoculating into 96-well plate, and placing into 5% CO 2 And placing the mixture in a constant temperature incubator at 37 ℃. After 24 hours, the wall-attached growth is carried out, the supernatant is discarded, and a blank control group and an 800nmol/L DD group are arranged. Each group had 6 duplicate wells and were removed after 72h incubation. PBS buffer was washed 3 times, 75% ethanol was fixed for 30min, PBS buffer was washed 3 times, 0.5% Triton X-100 was added, and the mixture was allowed to stand for 20min, buffer was washed 3 times, BSA was blocked for 20min, and Ki67 primary antibody (cell proliferation marker) and Nestin primary antibody (NSC marker) were added, respectively. Washing 3 times at 4 ℃ overnight with buffer solution, adding corresponding fluorescent secondary antibody, reacting for lh at 37 ℃, washing 3 times with buffer solution, counterstaining DAPI, and sealing with glycerol. Ki67 and Nestin biscationic cells were observed under a phase contrast inverted microscope and counted in photographs. Dihydroartemisinin Derivative (DD) group Ki67 and Nestin double cationsThe rate is significantly higher than that of the control group (p < 0.05). Example 3 Effect of Dihydroartemisinin derivatives on ischemic foci in rats with middle cerebral artery occlusion model Rat middle cerebral artery thrombosis establishes a rat MCAO model. The experimental animal is injected with chloral hydrate 5% into abdominal cavity, and the general anesthesia is cut through the front middle of neck, the right common carotid artery and carotid bifurcation are exposed and separated, a small opening is cut on the common carotid artery under the direct vision of ophthalmic scissors, a bolt wire is placed into the common carotid artery through the small opening to advance to the bifurcation of the middle cerebral artery, and the middle cerebral artery blood flow is obstructed. A negative Control group (postoperative daily intraperitoneal injection of 150mg/kg of physiological saline, control group), a positive Control group (postoperative daily intraperitoneal injection of aqueous solution of dihydroartemisinin (Dihydro artemisinin, DHA), 150mg/kg, DHA group) and an experimental group (postoperative daily intraperitoneal injection of aqueous solution of Dihydroartemisinin Derivative (DD) 150mg/kg, DD group) were set. Six animals per group. The animals were 7.0T cranium MR-examined 72h later and the examination results are shown in FIG. 4. And performing histogram analysis to find that: the volume of the cerebral infarction of the animals in the DD group is obviously lower than that of the Control group (p is less than 0.05), and the volume of the cerebral infarction of the animals in the DD group is obviously lower than that of the DHA group, but no statistical difference exists (p is more than 0.05). It is shown that the same dose of the Dihydroartemisinin Derivative (DD) has better effect than the dihydroartemisinin in treating cerebral infarction. After the three groups of animals are injected with the medicine, the animals move normally, no obvious toxicity appears, no death occurs after 14 days of continuous observation, and no obvious toxicity difference exists among the three groups. EXAMPLE 4 comparative study of Dihydroartemisinin derivatives across the blood brain barrier The rat middle cerebral artery occlusion model is established by the rat middle cerebral artery line embolism method. The experimental animal is injected with chloral hydrate 5% into abdominal cavity, and the general anesthesia is cut through the front middle of neck, the right common carotid artery and carotid bifurcation are exposed and separated, a small opening is cut on the common carotid artery under the direct vision of ophthalmic scissors, a bolt wire is placed into the common carotid artery through the small opening to advance to the bifurcation of the middle cerebral artery, and the middle cerebral artery blood flow is obstructed. A negative control group (daily intra-abdominal injection of physiological saline 150mg/kg after the operation), a positive control group (daily intra-abdominal injection of dihydroarteannuin aqueous solution (DHA) 150mg/kg after the operation) and an experimental group (daily intra-abdominal injection of dihydroarteannuin derivative aqueous solution (DD) 150mg/kg after the operation) were set, six animals per group. And taking a mouse brain tissue homogenate and a plasma sample after 72 hours, and detecting the concentration of the injection medicine respectively contained in the homogenate brain tissue and the plasma sample by using a high performance liquid chromatography. The plasma concentration of the dihydroartemisinin derivative group is not significantly different from that of the dihydroartemisinin group (p is more than 0.05). The brain tissue drug concentration of the dihydroartemisinin derivative group is obviously higher than that of the equivalent dose dihydroartemisinin group (p is less than 0.05). The blood brain barrier permeability of the same dose DD is increased by 40% compared with DHA, and according to the measurement, compared with DHA and DD, the same neuroprotective effect is achieved, and the dosage can be reduced by 30%. Claims (10) Hide Dependent 1. A dihydroartemisinin derivative, which has the structural formula: 2. a medicament comprising a dihydroartemisinin derivative of claim 1. 3. The medicament of claim 2, further comprising one or more pharmacologically acceptable excipients including diluents, excipients, fillers, wetting agents, absorption promoters, surfactants, lubricants or stabilizers and the like conventional in the pharmaceutical arts. 4. The medicament of claim 2, which is an injection. 5. The medicament of claim 4, wherein the injection is an aqueous solution with a medicament concentration of 150mg/kg. 6. Use of a dihydroartemisinin derivative according to claim 1 or a medicament according to any one of claims 2 to 5 in the treatment of, or prophylaxis of, cerebrovascular disease. 7. Use of a dihydroartemisinin derivative according to claim 1 or a medicament according to any one of claims 2 to 5 for the preparation of a medicament for the treatment and prevention of ischemic stroke. 8. Use of a dihydroartemisinin derivative according to claim 1 or a medicament according to any one of claims 2 to 5 for the preparation of a medicament for the alleviation or elimination of cerebral infarction ischemic events. 9. Use of a dihydroartemisinin derivative according to claim 1 or a medicament according to any one of claims 2 to 5 for the preparation of a medicament for promoting proliferation of neural stem cells. 10. Use of a dihydroartemisinin derivative according to claim 1 or a medicament according to any one of claims 2 to 5 for the preparation of a medicament for promoting proliferation of neural stem cells in the subventricular zone of the ventricle. 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CN202110514361.8A2021-05-11Dihydroartemisinin derivative and application thereof in preparation of drugs for treating and preventing cerebrovascular diseases https://patents.google.com/patent/CN116120337A/en 一种双氢青蒿素衍生物、药物及其在制备治疗预防脑血管病药物中的应用 技术领域 本发明涉及一种双氢青蒿素衍生物，并涉及其药物用途。 背景技术 随着我国老龄化进程，脑血管病已成为致死致残第一病因。主要病因是由于脑血管栓塞或者血栓形成所导致的血流中断，而造成神经细胞缺血缺氧死亡，病情严重程度与脑组织梗死体积密切相关。 神经干细胞(Neural Stem Cells，NSCs)主要存在于侧脑室壁的室管膜下区和海马的齿状回区，是一群可以自我更新、干性维持和分化为有功能神经元的特殊细胞。由于神经元为终末分化细胞，无法自我更新，其缺血死亡后只能通过神经干细胞增殖分化予以修复功能，而脑血管病研究中如何激活内源性神经干细胞快速增殖以修复梗死脑组织一直是热点领域。 本课题组一直致力于青蒿素及其在治疗神经性相关疾病的应用。 CN104523679A提供了双氢青蒿素在治疗、脑血管病的用途。虽然双氢青蒿素被发现对神经干细胞增殖有促进作用，体外可诱导神经干细胞增殖，体内可以减少脑梗塞的缺血灶。但是，研究表明长期低剂量的双氢青蒿素可能导致骨髓抑制、外周血红细胞减少和脾脏髓外造血等副反应，而减少剂量可以有效避免相关副反应的发生[尹纪业.两种青蒿素衍生物毒理学再评价及其心脏毒性机制研究[D].中国人民解放军军事医学科学院,2014.]。另外随着研究的深入发现，双氢青蒿素属于水溶性药物，虽可通过血脑屏障，但透过率有限，需要较高的血药浓度才能达到神经保护所需的脑组织浓度[Yepu He et al,Synergistic integration of dihydro-artemisinin withγ-aminobutyric acidresults in a more potential anti-depressant.Bioorg Chem.2021.110:104769.]。综上，寻找一种双氢青蒿素衍生物可达到更高的血脑屏障通透率，对减少其治疗脑血管病中产生的副反应具有重要临床意义。 发明内容 基于此问题，经过潜心研究，本发明的目的在于提供了一种新的双氢青蒿素衍生物，具备治疗预防脑血管病功能，改善了双氢青蒿素血脑屏障透过率低的问题，提高了脑组织中的药物浓度。 本发明的目的是通过以下措施实现的： 一种双氢青蒿素衍生物，其结构式为： 本发明还提供了一种药物。 一种药物，包括上述双氢青蒿素衍生物。所述药物还可以包括一种或多种药理学上可接受的辅料，所述辅料包括药学领域中常规的稀释剂、赋形剂、填充剂、湿润剂、吸收促进剂、表面活化剂、润滑剂或稳定剂等。所述药物优选制成水溶液或粉剂等适用于注射用的药物形式；更优选为水溶液，药物浓度为150mg/kg。所述药物在使用时可静脉滴注或肌肉注射，一般1天应用1次，每次40mg，14天为一个疗程。较双氢青蒿素常规剂量(60mg)减少30％用量。 本发明的另一目的在于提供上述双氢青蒿素衍生物的用途。 上述双氢青蒿素衍生物在治疗、预防脑血管病药物中的应用。进一步的，所述双氢青蒿素衍生物在制备治疗、预防缺血性脑卒中药物中的应用。更进一步的，所述双氢青蒿素衍生物在制备减轻或消除脑梗塞缺血灶药物中的应用。 上述双氢青蒿素衍生物在制备促进神经干细胞增殖药物中的应用。进一步地，所述双氢青蒿素衍生物促进大脑室管膜下区(Subventricular Zone，SVZ)神经干细胞(Neural Stem Cells，NSCs)增殖的应用。 所述双氢青蒿素衍生物对神经干细胞增殖有促进作用；体外可诱导神经干细胞增殖；体内可有效减少脑梗塞的缺血灶梗死灶并改善脑卒中造成的神经功能缺损。以所述双氢青蒿素衍生物为活性成分可制备成治疗缺血性脑卒中的药物，而且所述药物更容易通过血脑屏障，具有更高的脑组织药物浓度。 有益效果 1.脑缺血发生后，由于神经元对血氧的特殊依耐性，短期内就会有大量的有功能神经细胞死亡，给预后带来极其不利的影响。本发明所提供的双氢青蒿素衍生物，其保持了双氢青蒿素治疗神经性相关疾病的相关功能，具体而言，可促进内源性NSCs在缺血区神经细胞受到损伤后激活、增殖、分化为神经前体细胞(Neuroblasts)并向损伤灶迁移，在损伤的神经网络系统中重新整合，修复损伤的神经网络结构并可替代坏死神经元发挥相应的神经功能。所述双氢青蒿素衍生物通过早期激活(图2)神经干细胞内源性NSCs的增殖替代缺血灶周围坏死神经元而发挥功能，可用于脑梗塞的治疗。 2.本发明所提供的双氢青蒿素衍生物，不仅具有所述药效，而且更优的是较双氢青蒿素具有更优的血脑屏障透过率(图4)，可以对于中枢神经系统疾病具有更高的药物浓度，能够有效的减少药物用量，提高药物疗效，并进一步降低毒、副作用。 附图说明 图1实施例1中双氢青蒿素衍生物(Dihydroartemisinin derivatives，DD)制备中化学结构变化 图2实施例2免疫荧光结果显示：DD组的NSC增殖数量(Nestin/Ki67双染)较对照组明显增加； 图3实施例3中MCAO动物手术72h后的MRI照片，可见DD组动物脑梗死灶较对照组与双氢青蒿素(Dihydroartemisinin,DHA)组显著缩小 图4实施例4中MCAO动物手术72h后的高效液相色谱法测定血浆与脑组织药物浓度；A.血浆中DHA测定色谱图；B.脑组织中DD测定色谱图；C.血浆中浓度对比两组无显著差异；D.脑组织中浓度对比DD显著高于DHA。 具体实施方式 下面结合附图和实施例对本发明做进一步详细说明。以下实施例仅限于说明本发明而不用于限制本发明的范围。 在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属技术人员的理解相同的含义。若存在矛盾，本说明书优先。 下面将结合实施例对本发明的实施方式进行详细描述，实施例中未注明具体条件的，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商的，为可以通过商购获得的常规产品。 实施例1一种双氢青蒿素衍生物(Dihydroartemisinin derivatives，DD)的制备 1.10β-(2-溴乙氧基)双氢青蒿素的制备 将3.103g(24毫摩尔)2-溴乙基醇和100mL四氢呋喃加入到250mL的圆底烧瓶中，然后在冰浴条件下加入4mL BF3.Et2O(C4H10BF3O，三氟化硼乙醚溶液，商业购买)，再加入5.690g(20毫摩尔)的双氢青蒿素(C15H24O5)。该混合物在冰浴中搅拌反应1.5小时。反应过程用TLC监测。反应完成，分别加入饱和NaHCO3溶液和50mL EtOAc(CH3COOC2H5)，萃取分层后，将水层用EtOAc(30mL×2)萃取，然后将有机层合并。将有机层用40mL饱和盐水溶液洗涤，无水固体Na2SO4干燥，过滤后，减压旋转蒸馏有机溶剂。将所得到的粗产物重结晶(将石油醚滴加到溶解有样品EtOAc中)，静置，过滤，真空干燥，得白色晶体。 2.双氢青蒿素衍生物的制备 10β-(2-溴乙氧基)双氢青蒿素，CH3CN，KI，K2CO3和呋喃苄胺(Sigma-Aldrich公司商业购买)分别加入到一个100mL的圆底烧瓶中，该混合物反应在受控的温度下(50℃)加热反应。反应过程通过TLC监测。然后加入15mL二氯甲烷和20mL饱和NaCl溶液。萃取分层后，水层用二氯甲烷(10mL×2)萃取，然后将有机层合并。将有机层用20mL饱和盐水溶液洗涤，无水固体Na2SO4干燥；过滤后，减压旋转蒸馏二氯甲烷。硅胶柱色谱法(石油醚混合EtOAc)后，获得纯的产物。化合物结构变化过程如图1。 在Agilent核磁共振仪600MHz和151MHz条件下分别得到1H谱和13C谱，利用WatersSYNPAT G2仪器完成高分辨质谱测定所述化合物，谱图信息如下：1H NMR(600MHz,DMSO)d7.75(s,1H),6.57(s,1H),6.52(s,1H),5.40(s,1H),4.71(s,1H),4.20(s,2H,),3.89(s,1H),3.55(s,1H),3.08(s,2H),2.42(s,1H),2.18(s,1H),2.01(s,1H),1.80(s,1H),1.66(m,2H),1.54(s,1H),1.32(m,6H),1.14(s,1H),0.88(m,7H).13C NMR(151MHz,DMSO)d 146.84,143.96,111.41,110.87,103.43,101.06,86.89,80.34,63.65,51.90,45.75,43.83,42.83,36.46,36.01,34.09,30.27,25.62,24.22,23.95,20.11,12.62.HRMS m/z[M+H]+:408.2375(Calcd for C22H33NO6:407.230788)。 实施例2一种双氢青蒿素衍生物(DD)在离体条件下对神经干细胞增殖的影响 选取孕14.5d SD大鼠，5％水合氯醛腹腔麻醉，打开腹膜，取出胚胎，去除头皮、头骨和硬膜，剥离鼠脑。在解剖显微镜下，以嗅球为中心矢状位纵切，于室管膜下区取材。加入增殖培养基(DMEM/F12，bFGF 20ng/mL，EGF 20ng/mL，2％B27)，经200目滤网过滤后，台盼蓝染色后细胞计数，调整细胞数为1×106/ml，转入培养瓶，放入5％CO2、37℃恒温孵箱中培养，三天传代一次。 取对数期生长的NSCs，0.25％EDTA胰蛋白酶消化2min，10％DMEM/F12终止消化后，调整细胞数至8000/100μL，接种于的96孔板，置于5％CO2、37℃恒温培育箱中。24h后贴壁生长，弃上清，设置空白对照组、800nmol/L DD组共两组。每组6个复孔，孵育72h后取出。PBS缓冲液清洗3次，75％乙醇固定30min，PBS缓冲液清洗3次，加入0.5％的Triton X-100，静置20min，缓冲液洗3次，BSA封闭20min，分别加入Ki67一抗(细胞增殖标志物)与Nestin一抗(NSC标志物)。4℃过夜，缓冲液洗3次，加入相应的荧光二抗，37℃下反应lh，用缓冲液洗3次，复染DAPI，甘油封片。在相差倒置显微镜下观察Ki67与Nestin双阳性细胞并拍照计数。双氢青蒿素衍生物(DD)组Ki67与Nestin双阳性率明显高于对照组(p＜0.05)。 实施例3一种双氢青蒿素衍生物对大脑中动脉闭塞模型大鼠缺血灶的影响 大鼠大脑中动脉线栓法建立大鼠MCAO模型。实验动物5％水合氯醛腹腔注射全麻，经颈前正中切口、暴露并分离右侧颈总动脉及颈动脉分叉，眼科剪直视下于颈总动脉剪一小口，将栓线经此小口置入颈总动脉内向前推进至大脑中动脉分叉发出处，梗阻大脑中动脉血流。设置阴性对照组(术后每日腹腔注射生理盐水150mg/kg，Control组)，阳性对照组(术后每日腹腔注射双氢青蒿素水溶液(Dihydro artemisinin,DHA),150mg/kg，DHA组)和实验组(术后每日腹腔注射双氢青蒿素衍生物水溶液(DD)150mg/kg，DD组)。每组六只动物。72h后行小动物7.0T头颅MR检查，检查结果如图4。并做柱状图分析发现：DD组动物脑梗死体积显著低于Control组(p＜0.05)，且DD组动物脑梗死体积明显低于DHA组，但无统计学差异(p＞0.05)。说明同样剂量治疗脑梗死，双氢青蒿素衍生物(DD)较双氢青蒿素具有更好效果。 以上三组动物在注射药物后，活动正常，无明显毒性呈现，连续观察14天无动物发生死亡，三组间无明显的毒性差异。 实施例4一种双氢青蒿素衍生物透过血脑屏障的对比研究 大鼠大脑中动脉线栓法建立大鼠大脑中动脉阻塞模型。实验动物5％水合氯醛腹腔注射全麻，经颈前正中切口、暴露并分离右侧颈总动脉及颈动脉分叉，眼科剪直视下于颈总动脉剪一小口，将栓线经此小口置入颈总动脉内向前推进至大脑中动脉分叉发出处，梗阻大脑中动脉血流。设置阴性对照组(术后每日腹腔注射生理盐水150mg/kg)，阳性对照组(术后每日腹腔注射双氢青蒿素水溶液(Dihydroartemisinin,DHA),150mg/kg)和实验组(术后每日腹腔注射双氢青蒿素衍生物水溶液(DD)150mg/kg)，每组六只动物。72h后取小鼠脑组织匀浆与血浆标本，利用高效液相色谱法检测匀浆脑组织与血浆标本中分别所含注射药物的浓度。双氢青蒿素衍生物组血浆药浓度与双氢青蒿素组对比无显著差异(p＞0.05)。双氢青蒿素衍生物组脑组织药浓度明显高于等剂量双氢青蒿素组(p＜0.05)。同样剂量DD较DHA增加40％的血脑屏障通透率，据此测算，较DHA，DD达到同样神经保护效应，可减少30％用药量。 Claims (10) 1.一种双氢青蒿素衍生物，其结构式为： 2.一种药物，包括权利要求1所述的双氢青蒿素衍生物。 3.如权利要求2所述的药物，还包括一种或多种药理学上可接受的辅料，所述辅料包括药学领域中常规的稀释剂、赋形剂、填充剂、湿润剂、吸收促进剂、表面活化剂、润滑剂或稳定剂等。 4.如权利要求2所述的药物，所述药物为注射剂。 5.如权利要求4所述的药物，所述注射剂为水溶液，药物浓度为150mg/kg。 6.如权利要求1所述双氢青蒿素衍生物或权利要求2-5任一所述药物在治疗、预防脑血管病药物中的应用。 7.如权利要求1所述双氢青蒿素衍生物或权利要求2-5任一所述药物在制备治疗、预防缺血性脑卒中药物中的应用。 8.如权利要求1所述双氢青蒿素衍生物或权利要求2-5任一所述药物在制备减轻或消除脑梗塞缺血灶药物中的应用。 9.如权利要求1所述双氢青蒿素衍生物或权利要求2-5任一所述药物在制备促进神经干细胞增殖药物中的应用。 10.如权利要求1所述双氢青蒿素衍生物或权利要求2-5任一所述药物在制备促进大脑室管膜下区神经干细胞增殖药物中的应用。 Patent Citations (7) Publication numberPriority datePublication dateAssigneeTitle CN105732654A *2016-01-292016-07-06暨南大学二氢青蒿素-美金刚二联体化合物及其合成方法和用途 CN106588949A *2016-12-092017-04-26新乡学院一种具有心脑血管疾病防治活性的青蒿素酯类化合物、其制备方法及其应用 CN110642869A *2019-09-262020-01-03西南大学二氢青蒿素的硫醚、亚砜与砜衍生物及其应用 CN111747967A *2020-08-142020-10-09广州药本君安医药科技股份有限公司二氢青蒿素/神经递质拼合物及其合成方法和用途 CN113264946A *2021-05-112021-08-17中国人民解放军陆军军医大学第一附属医院一种双氢青蒿素衍生物及其在制备治疗、预防脑血管病药物中的应用 Family To Family Citations CN104523679A *2015-01-092015-04-22中国人民解放军第三军医大学第一附属医院青蒿琥酯在制备治疗、预防中枢神经损伤药物中的应用 WO2019104247A1 *2017-11-212019-05-31Ming 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CN202110514361.8A2021-05-11一种双氢青蒿素衍生物及其在制备治疗、预防脑血管病药物中的应用

The invention provides a method for increasing artemisinin content in sweet wormwood by DBR2 (double bond reductase 2) gene transfer in the field of biotechnology. The method includes the steps: cloning genes of artemisinic aldehyde delta 11(13) DBR2 from the sweet wormwood, constructing a plant expression vector containing the DBR2 genes, using agrobacterium tumefaciens for mediation to transfer the DBR2 genes into the sweet wormwood so that plants regenerate, using PCR (polymerase chain reaction) to detect integration conditions of the exogenous targeted DBR2 genes, determining the artemisinin content in the transgenic sweet wormwood by means of HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detector), and screening the transgenic sweet wormwood plants with the improved artemisinin content. The artemisinin content in the obtained transgenic sweet wormwood can be remarkably increased and is 2.83 times maximally of that of a non-transgenic control plant, and accordingly the method for increasing the artemisinin content in the sweet wormwood lays a foundation for large-scale artemisinin production by the aid of the transgenic sweet wormwood. 本发明是一种生物技术领域的转DBR2基因提高青蒿中青蒿素含量的方法。本发明从青蒿中克隆青蒿醛Δ11(13)还原酶DBR2的基因，构建含DBR2基因的植物表达载体，用根癌农杆菌介导，将DBR2基因转入青蒿并再生出植株，PCR检测外源目的基因DBR2的整合情况，高效液相色谱法及蒸发光散射检测器测定(HPLC-ELSD)转基因青蒿中青蒿素的含量，筛选获得青蒿素含量提高的转基因青蒿植株。本发明获得的转基因青蒿中青蒿素的含量显著提高，最高达到非转化对照植株的2.83倍，从而提供了一种提高青蒿中青蒿素含量的方法，为利用转基因青蒿大规模生产青蒿素打下了基础。 Description Turn the method that DBR2 gene improves content of artemisinin in sweet wormwood Technical field What the present invention relates to is a kind of method of raising artemislnin content of biological technical field, particularly a kind ofly turns the method that DBR2 gene improves content of artemisinin in sweet wormwood. Background technology Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Artemisinin (artemisinin) is a kind of sesquiterpene lactones compound containing peroxide bridge structure be separated from its over-ground part, be the medicine of the most effectively treating malaria of generally acknowledging in the world at present, particularly for encephalic malaria and anti-chloroquine malaria, there is quick-acting and feature that is low toxicity.At present, the method for the most effectively treating malaria of world health organisation recommendations is exactly Artemisinin conjoint therapy (ACTs).In addition, along with progressively going deep into Artemisinin pharmacological research, scientist finds that Artemisinin and derivative thereof also have anti-inflammatory, schistosomicide, antitumor and immunoregulatory function.Visible Artemisinin is a kind of natural drug of great potential. The main source of current Artemisinin extracts from the over-ground part of sweet wormwood plant, but the content of Artemisinin in Artemisia annuna is very low, in different planting environment and varieties of plant, its average content is at the 0.01-1% of sweet wormwood leaf dry weight, the large-scale commercial of this medicine is produced and is restricted.Due to Artemisinin complex structure, synthetic difficulty is large, and yield poorly, cost is high, does not have feasibility.The method of someone trial tissue culture and cell engineering produces Artemisinin, however Artemisinin in callus, content is lower than 0.1% of dry weight, the highest in bud also only have 0.16% of dry weight, and great majority research does not detect Artemisinin in root.Therefore utilize tissue culture and cell engineering not high to the feasibility of producing Artemisinin yet. Through finding prior art literature search, Waleerat Banyai etc. are in " Plant Cell Tissue and OrganCulture " (plant cell tissue's organ culture), within 2010,103 volume 255-265 pages have delivered the paper being entitled as " Overexpressionof farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth ofArtemisia annua L. " (" overexpression farnesyl pyrophosphate synthase gene can affect the content of Artemisinin in Artemisia annuna and the growth of sweet wormwood "), report by crossing most representation farnesyl pyrophosphate synthase (farnesyl pyrophosphatesynthase, FPS), the content of render transgenic Artemisinin in Artemisia annuna improves 2.5-3.6 doubly, but still only have about 1.3%.But, plant genetic engineering is improve the content of Artemisinin in Artemisia annuna to provide a feasible method. Sweet wormwood aldehyde Δ 11 (13) reductase enzyme (artemisinic aldehyde Δ 11 (13) double bondreductase in prior art, DBR2) being a key enzyme in artemisinin synthesis approach, is the important target spot of Artemisinin metabolic engineering.Adopt genetic engineering means, transform sweet wormwood with key gene DBR2, the biosynthetic speed limit bottleneck of Artemisinin will be broken, obtain the sweet wormwood plant of Artemisinin high yield, for large-scale production Artemisinin provides a new way. Summary of the invention The object of the invention is to overcome deficiency of the prior art, provide a kind of and turn the method that DBR2 gene improves content of artemisinin in sweet wormwood.The key gene clone that the present invention relates to, vector construction, genetic transformation, Molecular Detection, Artemisinin extraction and assay are for the present invention, establishing the stable method improving content of artemisinin in sweet wormwood, establishing solid basis for utilizing sweet wormwood scale operation Artemisinin. The present invention is achieved by the following technical solutions: Turn the method that DBR2 gene improves content of artemisinin in sweet wormwood, comprise following concrete steps: (1) gene clone method is adopted to obtain sweet wormwood key gene DBR2; (2) described DBR2 gene is linked to expression regulation sequence, builds the plant expression vector containing DBR2 gene; (3) by the described plant expression vector transform Agrobacterium tumefaciens containing DBR2 gene, the Agrobacterium tumefaciens strain containing described DBR2 gene plant expression vector is obtained; (4) utilize the described Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector to transform sweet wormwood, obtain the transgene abrotanum plant of the integration external source goal gene DBR2 detected through PCR; (5) carry out HPLC-ELSD mensuration to artemislnin content in described transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves. Preferably, in described step (1), described gene clone method comprises the following steps: extract sweet wormwood genome total serum IgE, obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL reverse transcription, DNA sequence dna according to SEQ ID NO.1, design amplifies upstream primer and the downstream primer of complete encoder block, described upstream primer is the DNA sequence dna shown in SEQ ID NO.2, described downstream primer is the DNA sequence dna shown in SEQ ID NO.3, and restriction endonuclease sites is introduced respectively so that construction of expression vector on described upstream and downstream primer, with the first described chain cDNA for template, check order after pcr amplification. Preferably, in described step (2), described structure comprises the following steps containing the plant expression vector of DBR2 gene: first build intermediate carrier pMDT18-DBR2, intermediate carrier pMDT18-DBR2 and expression vector FSN is cut again with XmaI and SacI enzyme, reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extract plasmid and do PCR detection and digestion verification. Preferred further, described structure intermediate carrier pMDT18-DBR2 comprises following concrete steps: by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme. Preferably, in described step (4), described conversion comprises the following steps: the preculture of explant; The Dual culture of Agrobacterium and explant; The screening of resistance regeneration plant. Preferred further, described preculture comprises the following steps: seeds of southernwood 75% alcohol immersion 1min, 20min is soaked again with 20%NaClO, aseptic water washing 3-4 time, blots surface-moisture with aseptic thieving paper, is inoculated in the MS solid medium without hormone, 25 DEG C of illumination cultivation, can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming. Preferred further, described Dual culture comprises the following steps: forwarded to by described leaf explant in Dual culture substratum, drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast. Preferred further, described screening comprises the following steps: be transferred in germination screening culture medium the described Dual culture sweet wormwood explant of 3 days in 25 DEG C of illumination cultivation, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, described well-grown resistance Multiple Buds is cut proceed to root media is cultured to and take root, Kan resistance regeneration sweet wormwood plant can be obtained. Preferably, in described step (4), described PCR detects and comprises the following steps: the primer of design and synthesis DBR2 gene; Carry out DNA cloning; Viewed under ultraviolet radiation, if object band is positive, then this strain is described transgene abrotanum plant. Preferably, in described step (5), described HPLC-ELSD measures and comprises following condition: chromatographic column used is C-18 reverse phase silica gel post, moving phase select volume ratio be 70: 30 methyl alcohol: water, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 20 μ L, light scattering detector drift tube temperature 40 DEG C, scale-up factor is 7, nebulizer gas pressure 5bar. The method turning DBR2 gene raising content of artemisinin in sweet wormwood of the present invention; adopt gene engineering method; key gene DBR2 is imported in sweet wormwood plant; obtain the transgene abrotanum strain that artemislnin content significantly improves; the content turning DBR2 gene Artemisinin in Artemisia annuna can reach the 22.6mg/g of dry weight; be 2.83 times of non-transformed common sweet wormwood (8mg/g dry weight), this invention provides high yield for the large-scale production for Artemisinin, to stablize source new drugs significant. Accompanying drawing explanation Fig. 1 is the content detection result figure of Artemisinin in sweet wormwood plant of the present invention. Embodiment Below embodiments of the invention are elaborated: the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises. embodiment Step one, the clone of sweet wormwood DBR2 gene (1) extraction of sweet wormwood genome total serum IgE Get 100-200mg sweet wormwood young leaflet tablet, after liquid nitrogen flash freezer, grind with mortar rapidly, add and fill 1mL TRlzol (TRlzol Reagents, GIBCOBRL, USA) 1.5mL Eppendorf pipe in, fully after vibration, 5min is put in ambient temperatare, add 200 μ L chloroforms, use forced oscillation 15sec, after room temperature places 2-3min, 12,000rmp centrifugal 15min at 4 DEG C; Supernatant liquor (about 600 μ L) is sucked in clean 1.5mL Eppendorf pipe, adds isopyknic Virahol, put upside down mixing, after ambient temperatare puts 10min, 12,000rmp centrifugal 10min at 4 DEG C; Abandon supernatant, add 1mL 75% ethanol purge, after vibration, 7,500rmp centrifugal 5min at 4 DEG C; Be dissolved in appropriate (30-40 μ L) RNAase-free water after drying at room temperature 10-15min; By denaturing formaldehyde gel electrophoresis qualification total serum IgE quality, then on spectrophotometer, measure rna content. (2) clone of sweet wormwood DBR2 gene Obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL (AMV) reverse transcription, according to the encoding sequence (DNA sequence dna shown in SEQ ID NO.1) of described sweet wormwood DBR2 gene, design amplifies upstream primer (DNA sequence dna shown in SEQ ID NO.2) and the downstream primer (DNA sequence dna shown in SEQ ID NO.3) of complete encoder block, and on upstream and downstream primer, introduce restriction endonuclease sites (this is determined by the carrier selected) respectively, so that construction of expression vector.With the first described chain cDNA for template, check order after pcr amplification.Determined dna sequence has been checked order by Shanghai Ying Jun biotechnology Services Co., Ltd.Sequencing result shows, the sequence of cloning is consistent with the encoding sequence (DNA sequence dna shown in SEQ ID NO.1) of the sweet wormwood DBR2 gene reported in GenBank. The present embodiment adopts gene clone method from sweet wormwood, obtain the correct Artemisinin biosynthesizing key gene DBR2 of sequence, for providing an important key gene by turning DBR2 gene raising content of artemisinin in sweet wormwood. Step 2, containing the structure of the plant binary expression vector of DBR2 gene (1) structure of intermediate carrier pMDT18-DBR2 Select pMDT18 carrier (Takara, Dalian) for primary element, build intermediate carrier pMDT18-DBR2.Particularly, by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme, being checked order by Shanghai Ying Jun biotechnology Services Co., Ltd confirms the exactness of gene. (2) containing the structure of the plant expression vector of DBR2 gene For expression vector, above-mentioned DBR2 gene is connected into the restriction enzyme site position of its correspondence with described FSN (FSN expression vector obtains for transforming on pCAMBIA2300 expression vector and preserves).Particularly, XmaI and SacI double digestion intermediate carrier pMDT18-dbr2 and expression vector FSN.Reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extract plasmid and do PCR detection and digestion verification. Artemisinin biosynthetic pathway key gene DBR2 is connected to expression regulation sequence by the present embodiment operably, and form the plant expression vector containing DBR2 gene, this expression vector can be used for the content being improved Artemisinin in Artemisia annuna by metabolic engineering strategies. Step 3, containing the acquisition of DBR2 gene double base plant expression vector agrobacterium tumefaciens engineering bacteria The above-mentioned plant binary expression vector containing DBR2 gene is proceeded to agrobacterium tumefaciens (as EHA105, for there is the biomaterial of public sale in market, can buy from Australian CAMBIA company, strain number is Gambar 1), and performing PCR of going forward side by side is verified. Step 4, Agrobacterium tumefaciens mediated DBR2 gene transformation sweet wormwood (1) preculture of explant Seeds of southernwood 75% alcohol immersion 1min, 20min is soaked again with 20%NaClO, aseptic water washing 3-4 time, surface-moisture is blotted with aseptic thieving paper, be inoculated in MS (the Murashige and Skoog without hormone, 1962), in solid medium, 25 DEG C, 16h/8h (light/dark) illumination cultivation, can obtain sweet wormwood aseptic seedling.Grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming. (2) Dual culture of Agrobacterium and explant By described leaf explant, forward in Dual culture substratum (1/2MS+AS 100 μm of ol/L), drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted, 28 DEG C of light culture 3 days with bacterium liquid.With drip the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene leaf explant for contrast. (3) screening of resistance regeneration plant The described Dual culture sweet wormwood explant of 3 days is transferred in germination screening culture medium (MS+6-BA 0.5mg/L+NAA 0.05mg/L+Kan 50mg/L+Cb 500mg/L) in 25 DEG C, 16h/8h (light/dark) illumination cultivation, every two weeks succeeding transfer culture once, can obtain Kan resistance Multiple Buds after 2-3 subculture.Well-grown resistance Multiple Buds is cut proceed to root media (1/2MS+Cb 125mg/L) is cultured to and take root, thus obtain Kan resistance regeneration sweet wormwood plant. Step 5, the PCR of transgene abrotanum plant detects Forward primer is designed respectively and reverse primer detects goal gene according to goal gene place expression cassette p35s-dbr2-nos sequence p35s and dbr2.Result shows, the PCR special primer designed by utilization, can amplify the specific DNA fragment of 458bp.And with non-transformed sweet wormwood genomic dna for template time, do not amplify any fragment. The present embodiment is by described plant expression vector transform Agrobacterium tumefaciens, obtain the Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector for transforming sweet wormwood, Agrobacterium tumefaciens strain constructed by utilization transforms sweet wormwood, obtains the transgene abrotanum plant detected through PCR.The acquisition of transgene abrotanum plant is screen the sweet wormwood strain obtaining higher artemislnin content to provide direct material. Step 6, utilizes HPLC-ELSD to measure artemislnin content in transgene abrotanum (1) preparation of HPLC-ELSD condition and system suitability and standardized solution HPLC: adopt water alliance 2695 system, chromatographic column is C-18 reverse phase silica gel post (Symmetry ShieldTM C18,5 μm, 250 × 4.6mm, Waters), moving phase is methyl alcohol: water, methyl alcohol: the volume ratio of water is 70: 30, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 10 μ L, sensitivity (AUFS=1.0), theoretical plate number calculates by Artemisinin peak and is not less than 2000. ELSD: adopt water alliance 2420 system, light scattering detector drift tube temperature 40 DEG C, scale-up factor (gain) is 7, nebulizer gas pressure 5bar; Precision takes Artemisinin standard substance (Sigma company) 2.0mg 1mL methyl alcohol and dissolves completely, obtains 2mg/mL Artemisinin standard solution, be stored in-20 DEG C for subsequent use. In the present embodiment, moving phase is methyl alcohol: water, and when ratio is 70%: 30%, the retention time of Artemisinin is 5.1min, and peak type is good.Theoretical plate number calculates by Artemisinin and is not less than 2000. (2) making of typical curve By described reference substance solution difference sample introduction 2 μ L under corresponding chromatographic condition, 4 μ L, 6 μ L, 8 μ L, 10 μ L record collection of illustrative plates and chromatographic parameters, carry out regression analysis respectively with peak area (Y) to standard substance content (X, μ g).By research, in the present embodiment, Artemisinin presents good log-log linear relationship within the scope of 4-20 μ g.The log-log equation of linear regression of Qinghaosu is Y=5.404e+0000X+1.858e+0000, R 2=0.999184. (3) preparation of sample and the mensuration of artemislnin content The leaching process of Artemisinin is based on the method reported in Van Nieuwerburgh et al. (2006): the sweet wormwood blade (1-2g fresh weight) taking a morsel fresh, be immersed in 50ml test tube in 10ml chloroform and swayed 1 minute, leach liquor is poured in new test tube and make chloroform volatilize completely, get 3ml dehydrated alcohol and fully dissolve extract, filter after filter filters through 0.22 μm and detect for HPLC.Meanwhile, 60 DEG C of baking ovens are put in the blade collection after chloroform extraction dries, weigh (dry weight calculating sweet wormwood blade); HPLC-ELSD is adopted to measure artemislnin content, sample feeding volume is 20 μ L, the artemislnin content (mg) in sample is gone out according to peak area substitution linear regression Equation for Calculating, again divided by artemisia leaf dry weight (g) of sample, thus calculate the content of Artemisinin in sweet wormwood plant. Turn DBR2 gene in an embodiment and significantly improve content of artemisinin in sweet wormwood.The content turning DBR2 gene blue or green senior middle school Artemisinin can reach the 22.6mg/g (as shown in Figure 1) of dry weight, is 2.83 times of non-transformed common sweet wormwood (8mg/g dry weight). The present embodiment adopts HPLC-ELSD method to determine artemislnin content in transgene abrotanum, adopts the metabolic engineering strategies transforming DBR2 gene to obtain the sweet wormwood plant of Artemisinin high yield, for large-scale production Artemisinin provides a kind of Perfected process. Claims (7) 1. turn the method that DBR2 gene improves content of artemisinin in sweet wormwood, it is characterized in that, comprise following concrete steps: (1) gene clone method is adopted to obtain sweet wormwood key gene DBR2; (2) described DBR2 gene is linked to expression regulation sequence, builds the plant expression vector containing DBR2 gene; (3) by the described plant expression vector transform Agrobacterium tumefaciens containing DBR2 gene, the Agrobacterium tumefaciens strain containing described DBR2 gene plant expression vector is obtained; (4) utilize the described Agrobacterium tumefaciens strain containing DBR2 gene plant expression vector to transform sweet wormwood, obtain the transgene abrotanum plant of the integration external source goal gene DBR2 detected through PCR; (5) carry out HPLC-ELSD mensuration to artemislnin content in described transgene abrotanum, screening obtains the transgene abrotanum plant that artemislnin content improves; In described step (1), described gene clone method comprises the following steps: extract sweet wormwood genome total serum IgE, obtained sweet wormwood genome total serum IgE is obtained the first chain cDNA by ThermoScript II XL reverse transcription, DNA sequence dna according to SEQ IDNO.1, design amplifies upstream primer and the downstream primer of complete encoder block, described upstream primer is the DNA sequence dna shown in SEQ ID NO.2, described downstream primer is the DNA sequence dna shown in SEQ ID NO.3, and restriction endonuclease sites is introduced respectively so that construction of expression vector on described upstream and downstream primer, with the first described chain cDNA for template, check order after pcr amplification, In described step (2), described structure comprises the following steps containing the plant expression vector of DBR2 gene: first build intermediate carrier pMDT18-DBR2, intermediate carrier pMDT18-DBR2 and expression vector FSN is cut again with XmaI and SacI enzyme, reclaim DBR2 gene fragment and FSN carrier large fragment, connect and transform, picking mono-clonal, extracts plasmid and does PCR detection and digestion verification; Described structure intermediate carrier pMDT18-DBR2 comprises following concrete steps: by introducing the full length gene of XmaI and SacI restriction enzyme site before and after high-fidelity enzymatic amplification DBR2 gene respectively, be connected on pMDT18 carrier by ligase enzyme. 2. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, it is characterized in that, in described step (4), described conversion comprises the following steps: the preculture of explant; The Dual culture of Agrobacterium and explant; The screening of resistance regeneration plant. 3. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described preculture comprises the following steps: seeds of southernwood 75% alcohol immersion 1min, then soaks 20min with 20%NaClO, aseptic water washing 3-4 time, surface-moisture is blotted with aseptic thieving paper, be inoculated in the MS solid medium without hormone, 25 DEG C of illumination cultivation, can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, clip tests for sterility explant is used for transforming. 4. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described Dual culture comprises the following steps: forwarded to by described leaf explant in Dual culture substratum, drip the 1/2MS suspension containing the described agrobacterium tumefaciens engineering bacteria containing DBR2 gene plant binary expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast. 5. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 2, it is characterized in that, described screening comprises the following steps: be transferred in germination screening culture medium the described Dual culture sweet wormwood explant of 3 days in 25 DEG C of illumination cultivation, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, described well-grown resistance Multiple Buds is cut proceed to root media is cultured to and take root, Kan resistance regeneration sweet wormwood plant can be obtained. 6. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, is characterized in that, in described step (4), described PCR detects and comprises the following steps: the primer of design and synthesis DBR2 gene; Carry out DNA cloning; Viewed under ultraviolet radiation, if object band is positive, then this strain is described transgene abrotanum plant. 7. the method turning DBR2 gene raising content of artemisinin in sweet wormwood according to claim 1, it is characterized in that, in described step (5), described HPLC-ELSD measures and comprises following condition: chromatographic column used is C-18 reverse phase silica gel post, and moving phase selects volume ratio to be the methyl alcohol of 70:30: water, column temperature 30 DEG C, flow velocity 1.0mL/min, sample size 20 μ L, light scattering detector drift tube temperature 40 DEG C, scale-up factor is 7, nebulizer gas pressure 5bar. https://patents.google.com/patent/CN102676578B/en 转DBR2基因提高青蒿中青蒿素含量的方法 技术领域 本发明涉及的是一种生物技术领域的提高青蒿素含量的方法，特别是一种转DBR2基因提高青蒿中青蒿素含量的方法。 背景技术 青蒿(Artemisia annua L.)是菊科蒿属的一年生草本植物。青蒿素(artemisinin)是从其地上部分分离的一种含有过氧桥结构的倍半萜内酯化合物，是目前世界上公认的最有效的治疗疟疾的药物，特别是对于脑型疟疾和抗氯喹疟疾具有速效和低毒的特点。目前，世界卫生组织推荐的最有效的治疗疟疾的方法就是青蒿素联合疗法(ACTs)。另外，随着对青蒿素药理研究的逐步深入，科学家发现青蒿素及其衍生物还具有抗炎、抗血吸虫、抗肿瘤以及免疫调节的功能。可见青蒿素是一种极具潜力的天然药物。 目前青蒿素的主要来源是从青蒿植株的地上部分提取，然而青蒿中青蒿素的含量非常低，不同种植环境和种植品种中其平均含量在青蒿叶片干重的0.01-1％，使得这种药物的大规模商业化生产受到了限制。由于青蒿素结构复杂，人工合成难度大，产量低，成本高，不具有可行性。有人尝试用组织培养和细胞工程的方法来生产青蒿素，然而青蒿素在愈伤组织中含量低于干重的0.1％，在芽中最高也只有干重的0.16％，而大多数研究在根中没有检测到青蒿素。因此利用组织培养及细胞工程来生产青蒿素的可行性也不高。 经对现有技术文献检索发现，Waleerat Banyai等在《Plant Cell Tissue and OrganCulture》(植物细胞组织器官培养)，2010年103卷255-265页发表了题为“Overexpressionof farnesyl pyrophosphate synthase(FPS)gene affected artemisinin content and growth ofArtemisia annua L.”(“过量表达法尼基焦磷酸合酶基因能影响青蒿中青蒿素的含量及青蒿的生长”)的论文，报道通过过最表达法尼基焦磷酸合酶(farnesyl pyrophosphatesynthase，FPS)，使转基因青蒿中青蒿素的含量提高了2.5-3.6倍，但仍然只有1.3％左右。不过，植物基因工程为提高青蒿中青蒿素的含量提供了一条可行的方法。 现有技术中青蒿醛Δ11(13)还原酶(artemisinic aldehydeΔ11(13)double bondreductase，DBR2)是青蒿素合成途径中的一个关键酶，是青蒿素代谢工程的重要靶点。采用基因工程手段，用关键酶基因DBR2转化青蒿，将打破青蒿素生物合成的限速瓶颈，获得青蒿素高产的青蒿植株，为规模化生产青蒿素提供一条新途径。 发明内容 本发明的目的在于克服现有技术中的不足，提供一种转DBR2基因提高青蒿中青蒿素含量的方法。本发明涉及的关键酶基因克隆、载体构建、遗传转化、分子检测、青蒿素提取及含量测定用于本发明，建立了稳定提高青蒿中青蒿素含量的方法，为利用青蒿大规模生产青蒿素奠定坚实的基础。 本发明是通过以下技术方案实现的： 一种转DBR2基因提高青蒿中青蒿素含量的方法，包括如下具体步骤： (1)采用基因克隆方法获得青蒿关键酶基因DBR2； (2)把所述DBR2基因连结于表达调控序列，构建含DBR2基因的植物表达载体； (3)将所述含DBR2基因的植物表达载体转化根癌农杆菌，获得含所述DBR2基因植物表达载体的根癌农杆菌菌株； (4)利用所述含DBR2基因植物表达载体的根癌农杆菌菌株转化青蒿，获得经PCR检测的整合外源目的基因DBR2的转基因青蒿植株； (5)对所述转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量提高的转基因青蒿植株。 优选的，在所述步骤(1)中，所述基因克隆方法包括以下步骤：提取青蒿基因组总RNA，将所获的青蒿基因组总RNA通过反转录酶XL反转录获得第一链cDNA，根据SEQ ID NO.1所示的DNA序列，设计扩增出完整编码框的上游引物和下游引物，所述上游引物为SEQ ID NO.2所示的DNA序列，所述下游引物为SEQ ID NO.3所示的DNA序列，并在所述上游和下游引物上分别引入限制性内切酶位点以便构建表达载体，以所述的第一链cDNA为模板，经PCR扩增后进行测序。 优选的，在所述步骤(2)中，所述构建含DBR2基因的植物表达载体包括以下步骤：先构建中间载体pMDT18-DBR2，再用XmaI和SacI酶切中间载体pMDT18-DBR2和表达载体FSN，回收DBR2基因片段和FSN载体大片段，连接转化，挑取单克隆，提取质粒做PCR检测和酶切验证。 进一步优选的，所述构建中间载体pMDT18-DBR2包括以下具体步骤：通过高保真酶扩增DBR2基因前后分别引入XmaI和SacI酶切位点的基因全长，通过连接酶连接到pMDT18载体上。 优选的，在所述步骤(4)中，所述转化包括以下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。 进一步优选的，所述预培养包括以下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃光照培养，即可获得青蒿无菌苗，待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。 进一步优选的，所述共培养包括以下步骤：将所述叶片外植体转到共培养培养基中，滴加含活化好的所述含DBR2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天，以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。 进一步优选的，所述筛选包括以下步骤：将所述共培养3天的青蒿外植体转入到发芽筛选培养基上于25℃光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将所述生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，即可获得Kan抗性再生青蒿植株。 优选的，在所述步骤(4)中，所述PCR检测包括以下步骤：设计合成DBR2基因的引物；进行DNA扩增；紫外线下观察，若目的条带为阳性，则该株系即为所述转基因青蒿植株。 优选的，在所述步骤(5)中，所述HPLC-ELSD测定包括以下条件：所用色谱柱为C-18反相硅胶柱，流动相选用体积比为70∶30的甲醇∶水，柱温30℃，流速1.0mL/min，进样量20μL，蒸发光散射检测器漂移管温度40℃，放大系数为7，载气压力5bar。 本发明的转DBR2基因提高青蒿中青蒿素含量的方法，采用基因工程方法，将关键酶基因DBR2导入青蒿植株中，获得了青蒿素含量显著提高的转基因青蒿株系，转DBR2基因青蒿中青蒿素的含量最高可达到干重的22.6mg/g，是非转化普通青蒿(8mg/g干重)的2.83倍，该发明对于为青蒿素的规模化生产提供高产、稳定新药源具有重要意义。 附图说明 图1为本发明的青蒿植株中青蒿素的含量检测结果图。 具体实施方式 下面对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。 实施例 步骤一，青蒿DBR2基因的克隆 (1)青蒿基因组总RNA的提取 取100-200mg青蒿幼嫩叶片，用液氮速冻后，迅速用研钵研碎，加入盛有1mL TRlzol(TRlzol Reagents，GIBCOBRL，USA)的1.5mL Eppendorf管中，充分振荡后，于室温下放置5min，加200μL氯仿，用力振荡15sec，室温放置2-3min后，于4℃下12,000rmp离心15min；将上清液(约600μL)吸入干净的1.5mL Eppendorf管中，加入等体积的异丙醇，颠倒混匀，室温下放置10min后，于4℃下12,000rmp离心10min；弃上清，加1mL 75％乙醇清洗，振荡后，于4℃下7,500rmp离心5min；室温干燥10-15min后溶于适量(30-40μL)RNAase-free水中；用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。 (2)青蒿DBR2基因的克隆 将所获的青蒿基因组总RNA通过反转录酶XL(AMV)反转录获得第一链cDNA，根据所述青蒿DBR2基因的编码序列(SEQ ID NO.1所示的DNA序列)，设计扩增出完整编码框的上游引物(SEQ ID NO.2所示的DNA序列)和下游引物(SEQ ID NO.3所示的DNA序列)，并在上游和下游引物上分别引入限制性内切酶位点(这可视选用的载体而定)，以便构建表达载体。以所述的第一链cDNA为模板，经PCR扩增后进行测序。DNA序列测定由上海英骏生物技术服务有限公司测序完成。测序结果表明，所克隆的序列与GenBank中所报道的青蒿DBR2基因的编码序列(SEQ ID NO.1所示的DNA序列)一致。 本实施例采用基因克隆方法从青蒿中获得序列正确的青蒿素生物合成关键酶基因DBR2，为通过转DBR2基因提高青蒿中青蒿素含量提供了一个重要关键酶基因。 步骤二，含DBR2基因的植物双元表达载体的构建 (1)中间载体pMDT18-DBR2的构建 选用pMDT18载体(Takara，Dalian)为基本元件，构建中间载体pMDT18-DBR2。具体地，通过高保真酶扩增DBR2基因前后分别引入XmaI和SacI酶切位点的基因全长，通过连接酶连接到pMDT18载体上，由上海英骏生物技术服务有限公司测序确认基因的正确性。 (2)含DBR2基因的植物表达载体的构建 以所述的FSN(FSN表达载体为在pCAMBIA2300表达载体上改造获得并保存)为表达载体，将上述DBR2基因连入其对应的酶切位点位置。具体地，XmaI和SacI双酶切中间载体pMDT18-dbr2和表达载体FSN。回收DBR2基因片段和FSN载体大片段，连接转化，挑取单克隆，提取质粒做PCR检测和酶切验证。 本实施例将青蒿素生物合成途径关键酶基因DBR2可操作性地连接于表达调控序列，形成含DBR2基因的植物表达载体，该表达载体可用于通过代谢工程策略来提高青蒿中青蒿素的含量。 步骤三，含DBR2基因双元植物表达载体根癌农杆菌工程菌的获得 将上述含DBR2基因的植物双元表达载体转入根癌农杆菌(如EHA105，为市场有公开出售的生物材料，可以从澳大利亚CAMBIA公司购得，菌株编号为Gambar 1)，并进行PCR验证。 步骤四，根癌农杆菌介导DBR2基因转化青蒿 (1)外植体的预培养 青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS(Murashige and Skoog，1962)固体培养基中，25℃、16h/8h(light/dark)光照培养，即可获得青蒿无菌苗。待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。 (2)农杆菌与外植体的共培养 将所述的叶片外植体，转到共培养培养基(1/2MS+AS 100μmol/L)中，滴加含活化好的所述含DBR2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天。以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。 (3)抗性再生植株的筛选 将所述的共培养3天的青蒿外植体转入到发芽筛选培养基(MS+6-BA 0.5mg/L+NAA 0.05mg/L+Kan 50mg/L+Cb 500mg/L)上于25℃、16h/8h(light/dark)光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽。将生长良好的抗性丛生芽剪下转入生根培养基(1/2MS+Cb 125mg/L)上培养至生根，从而获得Kan抗性再生青蒿植株。 步骤五，转基因青蒿植株的PCR检测 根据目的基因所在表达盒p35s-dbr2-nos序列p35s和dbr2分别设计正向引物和反向引物对目的基因进行检测。结果表明，利用所设计的PCR特异引物，能扩增出458bp的特异DNA片段。而以非转化青蒿基因组DNA为模板时，没有扩增出任何片段。 本实施例将所述的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含DBR2基因植物表达载体的根癌农杆菌菌株，利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测的转基因青蒿植株。转基因青蒿植株的获得为筛选获得较高青蒿素含量的青蒿株系提供了直接素材。 步骤六，利用HPLC-ELSD测定转基因青蒿中青蒿素含量 (1)HPLC-ELSD条件及系统适用性以及标准溶液的配制 HPLC：采用water alliance 2695系统，色谱柱为C-18反相硅胶柱(Symmetry ShieldTM C18，5μm，250×4.6mm，Waters)，流动相为甲醇∶水，甲醇∶水的体积比为70∶30，柱温30℃，流速1.0mL/min，进样量10μL，灵敏度(AUFS＝1.0)，理论塔板数按青蒿素峰计算不低于2000。 ELSD：采用water alliance 2420系统，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar； 精密称取青蒿素标准品(Sigma公司)2.0mg用1mL甲醇完全溶解，得到2mg/mL青蒿素标准品溶液，保存于-20℃备用。 本实施例中流动相为甲醇∶水，比例为70％∶30％时，青蒿素的保留时间为5.1min，峰型良好。理论塔板数按青蒿素计算不低于2000。 (2)标准曲线的制作 将所述对照品溶液在相应色谱条件下分别进样2μL，4μL，6μL，8μL，10μL记录图谱及色谱参数，分别以峰面积(Y)对标准品含量(X，μg)进行回归分析。通过研究，本实施例中青蒿素在4-20μg范围内呈现良好的log-log线性关系。青蒿素对照品的log-log线性回归方程为Y＝5.404e+0000X+1.858e+0000，R2＝0.999184。 (3)样品的制备和青蒿素含量的测定 青蒿素的提取过程基于Van Nieuwerburgh et al.(2006)中报道的方法：取少量新鲜的青蒿叶片(1-2g鲜重)，于50ml试管中将其浸没在10ml氯仿中摇荡1分钟，将浸出液倒入新的试管中使氯仿挥发完全，取3ml无水乙醇充分溶解提取物，经0.22μm过滤滤头过滤后用于HPLC检测。同时，氯仿提取后的叶片收集放入60℃烘箱进行烘干，称重(计算青蒿叶片的干重)； 采用HPLC-ELSD测定青蒿素含量，样品进样体积为20μL，根据峰面积代入线形回归方程计算出样品中的青蒿素含量(mg)，再除以样品的青蒿叶干重(g)，从而计算出青蒿植株中青蒿素的含量。 在实施例中转DBR2基因显著提高青蒿中青蒿素含量。转DBR2基因青高中青蒿素的含量最高可达到干重的22.6mg/g(如图1所示)，是非转化普通青蒿(8mg/g干重)的2.83倍。 本实施例采用HPLC-ELSD法测定了转基因青蒿中青蒿素含量，采用转化DBR2基因的代谢工程策略获得了青蒿素高产的青蒿植株，为规模化生产青蒿素提供了一种理想方法。 Claims (7) 1.一种转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，包括如下具体步骤： (1)采用基因克隆方法获得青蒿关键酶基因DBR2； (2)把所述DBR2基因连结于表达调控序列，构建含DBR2基因的植物表达载体； (3)将所述含DBR2基因的植物表达载体转化根癌农杆菌，获得含所述DBR2基因植物表达载体的根癌农杆菌菌株； (4)利用所述含DBR2基因植物表达载体的根癌农杆菌菌株转化青蒿，获得经PCR检测的整合外源目的基因DBR2的转基因青蒿植株； (5)对所述转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量提高的转基因青蒿植株； 在所述步骤(1)中，所述基因克隆方法包括以下步骤：提取青蒿基因组总RNA，将所获的青蒿基因组总RNA通过反转录酶XL反转录获得第一链cDNA，根据SEQ IDNO.1所示的DNA序列，设计扩增出完整编码框的上游引物和下游引物，所述上游引物为SEQ ID NO.2所示的DNA序列，所述下游引物为SEQ ID NO.3所示的DNA序列，并在所述上游和下游引物上分别引入限制性内切酶位点以便构建表达载体，以所述的第一链cDNA为模板，经PCR扩增后进行测序； 在所述步骤(2)中，所述构建含DBR2基因的植物表达载体包括以下步骤：先构建中间载体pMDT18-DBR2，再用XmaI和SacI酶切中间载体pMDT18-DBR2和表达载体FSN，回收DBR2基因片段和FSN载体大片段，连接转化，挑取单克隆，提取质粒做PCR检测和酶切验证；所述构建中间载体pMDT18-DBR2包括以下具体步骤：通过高保真酶扩增DBR2基因前后分别引入XmaI和SacI酶切位点的基因全长，通过连接酶连接到pMDT18载体上。 2.根据权利要求1所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，在所述步骤(4)中，所述转化包括以下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。 3.根据权利要求2所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，所述预培养包括以下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃光照培养，即可获得青蒿无菌苗，待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。 4.根据权利要求2所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，所述共培养包括以下步骤：将所述叶片外植体转到共培养培养基中，滴加含活化好的所述含DBR2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3天，以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。 5.根据权利要求2所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，所述筛选包括以下步骤：将所述共培养3天的青蒿外植体转入到发芽筛选培养基上于25℃光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将所述生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，即可获得Kan抗性再生青蒿植株。 6.根据权利要求1所述的转DBR2基因提高青蒿中青蒿素含量的方法，其特征是，在所述步骤(4)中，所述PCR检测包括以下步骤：设计合成DBR2基因的引物；进行DNA扩增；紫外线下观察，若目的条带为阳性，则该株系即为所述转基因青蒿植株。 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numberPriority datePublication dateAssigneeTitle Family To Family Citations CN105296536A *2015-11-122016-02-03上海交通大学一种转基因青蒿植株及其培育方法 CN105695507B *2016-04-272020-02-07上海交通大学转ics1基因提高青蒿中青蒿素含量的方法 CN106755060B *2016-11-172020-05-26上海交通大学共转fps和dbr2基因提高青蒿素含量的方法及制备的青蒿 CN107142287B *2017-05-122020-12-08南京林业大学青蒿醛双键还原酶DBR1及其重组菌在制备二氢-β-紫罗兰酮中的应用 CN110438148A *2019-07-242019-11-12中国人民解放军第二军医大学一种利用AaSPL2基因提高青蒿中青蒿素含量的方法 CN110628809A *2019-07-242019-12-31中国人民解放军第二军医大学一种利用AaTGA6基因提高青蒿中青蒿素含量的方法 CN114231655B *2021-12-292023-06-23华智生物技术有限公司一种与青蒿素含量连锁的snp位点及其应用 CN115918546B *2022-09-062024-05-07上海交通大学一种转基因青蒿以及提高青蒿中青蒿素含量的方法 * Cited by examiner, † Cited by third party, ‡ Family to family citation Similar Documents PublicationPublication DateTitle CN102676578B2015-07-08转dbr2基因提高青蒿中青蒿素含量的方法 CN101182544B2011-09-21转ads基因提高青蒿中青蒿素含量的方法 CN102776225A2012-11-14转AaWRKY1基因提高青蒿中青蒿素含量的方法 Benyammi et al.2016Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass 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A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene, include the following steps: to clone salicylic acid signal pathway AaSPL2 gene from sweet wormwood, construct the plant expression vector of the gene containing AaSPL2, with Agrobacterium tumefaciens mediated, AaSPL2 gene is transferred to sweet wormwood and regenerates plant, PCR detects the integration of external source target gene AaSPL2, then measures the content of qinghaosu in transgene abrotanum, and screening obtains the transgene abrotanum plant that artemislnin content improves.The content of qinghaosu significantly improves in the transgene abrotanum that the present invention obtains, up to the 1.8 of nontransformed control plant times, lays the foundation for qinghaosu is mass produced using transgene abrotanum. 一种利用AaSPL2基因提高青蒿中青蒿素含量的方法，包括如下步骤：从青蒿中克隆水杨酸信号途径AaSPL2基因，构建含AaSPL2基因的植物表达载体，用根癌农杆菌介导，将AaSPL2基因转入青蒿并再生出植株，PCR检测外源目的基因AaSPL2的整合情况，再测定转基因青蒿中青蒿素的含量，筛选获得青蒿素含量提高的转基因青蒿植株。本发明获得的转基因青蒿中青蒿素的含量显著提高，最高达到非转化对照植株的1.8倍，为利用转基因青蒿大规模生产青蒿素打下了基础。 Description A method of content of artemisinin in sweet wormwood is improved using AaSPL2 gene Technical field The invention belongs to field of biotechnology.A kind of method for improving artemislnin content is particularly related to, in particular to It is a kind of method for improving content of artemisinin in sweet wormwood using AaSPL2 gene. Background technique Sweet wormwood (Artemisia annua L.) is the annual herb plant of composite family artemisia.Qinghaosu (artemisinin) it is a kind of sesquiterpene lactone containing peroxide bridge structure separated from its aerial part, is current The drug of generally acknowledged most effective treatment malaria in the world has quick-acting and low especially for encephalic malaria and anti-chlorine quinoline malaria The feature of poison.Currently, the method for the most effective treatment malaria of world health organisation recommendations is exactly qinghaosu conjoint therapy (ACTs).In addition, it is anti-that scientist has found that qinghaosu and its derivative also have with gradually going deep into qinghaosu pharmacological research Inflammation, anti-schistosome, antitumor and immunoregulatory function.It can be seen that qinghaosu is a kind of natural drug of great potential. The main source of qinghaosu is extracted from the aerial part of sweet wormwood plant at present, however the content of Artemisinin in Artemisia annuna Very low (0.01%-1%), so that the large-scale commercial production of this drug is restricted.Since qinghaosu structure is multiple Miscellaneous, artificial synthesized difficulty is big, and low output is at high cost, does not have feasibility.Have tried to the side with tissue cultures and cell engineering Method produces qinghaosu, however qinghaosu content in callus is lower than the 0.1% of dry weight, and highest also only has dry weight in bud 0.16%, and most of research does not detect qinghaosu in root.Therefore it is produced using tissue cultures and cell engineering The feasibility of qinghaosu is not also high. Through to existing technical literature retrieve find, Qian Shen etc. " New Phytologist " (" new plant scholar ", 1269-1281 pages of 210 phase in 2016) deliver entitled " The jasmonate-responsive AaMYC2transcription factor positively regulates artemisinin biosynthesis in Artemisia annua " (" is closed by the biology that the AaMYC2 transcription factor that methyl jasmonic acid responds just is regulating and controlling Artemisinin in Artemisia annuna At ") paper, report mentions artemislnin content by being overexpressed the AaMYC2 that is responded by hormone methyl jasmonic acid in sweet wormwood It is 1.6 times high.Therefore, it is provided by being overexpressed the gene responded by methyl jasmonic acid to improve the content of Artemisinin in Artemisia annuna One feasible method. Summary of the invention The purpose of the present invention is to provide a kind of methods for improving content of artemisinin in sweet wormwood using AaSPL2 gene, will be green The AaSPL2 gene cloned in wormwood artemisia, which is transferred in sweet wormwood, obtains transgenosis AaSPL2 sweet wormwood, sweet wormwood in transgenosis AaSPL2 sweet wormwood Cellulose content significantly improves, and qinghaosu is mass produced using sweet wormwood to realize. The present invention is achieved by the following technical solutions: the present invention clones AaSPL2 gene from sweet wormwood, and building contains The plant expression vector of AaSPL2 gene, use is Agrobacterium tumefaciens mediated, by AaSPL2 channel genes sweet wormwood and regenerates plant； PCR detects the integration of external source target gene AaSPL2, HPLC ELSD detector (HPLC-ELSD) Content of artemisinin in sweet wormwood is measured, screening obtains the transgene abrotanum plant that artemislnin content improves. The method provided by the invention for improving content of artemisinin in sweet wormwood using AaSPL2 gene, includes the following steps: S1: sweet wormwood key gene AaSPL2 is obtained using gene clone method； S2: AaSPL2 gene is operatively connectable to expression regulation sequence, constructs the plant table of the gene containing AaSPL2 Up to carrier； S3: the plant expression vector of the gene containing AaSPL2 is converted into Agrobacterium tumefaciems, is obtained for converting containing for sweet wormwood The Agrobacterium tumefaciens strain of AaSPL2 gene plant expression vector； S4: sweet wormwood is converted using constructed Agrobacterium tumefaciens strain, obtains the transgene abrotanum through PCR test positive Plant； S5: HPLC-ELSD measurement is carried out to artemislnin content in the transgene abrotanum of acquisition, screening obtains artemislnin content The transgene abrotanum plant significantly improved. Preferably, step S1: sweet wormwood genome total serum IgE is extracted；Sweet wormwood genome total serum IgE is obtained first by reverse transcription Chain cDNA；According to the design of the coded sequence of the sweet wormwood AaSPL2 gene as shown in SEQ ID NO.1 amplifies complete encoder block Upstream and downstream primer, and restriction endonuclease sites are introduced respectively on upstream and downstream primer, so as to construction of expression vector；With institute Stating the first chain cDNA is template, is sequenced after PCR amplification, obtains and correct sweet wormwood AaSPL2 gene is sequenced, and wherein PCR expands The nucleotide sequence for increasing upstream and downstream primer used is respectively as follows: AaSPL2-PF:atggagtggaattgggacaa, AaSPL2- PR:ttaatttgaacaaaagtagtcaaaa. Preferably, in step S2, the plant expression vector of building gene containing AaSPL2 includes the following steps: to select PBI121 and pCAMBIA2300 is primary element, constructs binary plant expression vector pCAMBIA2300::p35S-gus-nos； BamHI and SacI double digestion carrier pCAMBIA2300::p35S-gus-nos is used again, recycles pCAMBIA2300::p35S-nos Large fragment will be formed by seamless Cloning Kit in AaSPL2 gene swapping to pCAMBIA2300::p35S-nos PCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos, conversion, picking monoclonal extract plasmid and do PCR detection and digestion verification. It is furthermore preferred that the building binary plant expression vector pCAMBIA2300::p35S-gus-nos includes following step It is rapid: to use HindIII and EcoRI double digestion pBI121 and pCAMBIA2300 plasmid；Recycle pBI121 gus expression cassette and PCAMBIA2300 large fragment；Recovery product is connected, transformation and selection takes out plasmid enzyme restriction verifying. Preferably, described to convert the preculture for including the following steps: explant in step S4；Agrobacterium and explant It co-cultures；The screening of resistance regeneration plant. It is furthermore preferred that the preculture of the explant includes the following steps: that 75% ethyl alcohol of seeds of southernwood impregnates 1min, then 20min is impregnated with 20%NaClO, aseptic water washing 3-4 times blots surface moisture with sterile blotting paper, is inoculated in no hormone In MS solid medium, illumination cultivation 16h, dark culture 8h, can be obtained sweet wormwood aseptic seedling at 25 DEG C, long to 5~8cm to seedling Afterwards, clip tests for sterility explant is for converting. It is furthermore preferred that the co-cultivation of the Agrobacterium and explant includes the following steps: the tests for sterility explant Body is gone in co-culture medium, is added dropwise containing the activated binary expression vector of gene plant containing AaSPL2 The 1/2MS of the Agrobacterium tumefaciems engineering bacteria of pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos is outstanding Liquid comes into full contact with explant with bacterium solution, 28 DEG C of dark culture 3d. It is furthermore preferred that include the following steps: will be outside the sweet wormwood of the described co-cultivation 3d for the screening of the resistance regeneration plant Implant is transferred on germination screening and culturing medium illumination cultivation 16h, dark culture 8h at 25 DEG C, and squamous subculture is primary every two weeks, warp It can be obtained Kan resistance Multiple Buds after crossing 2-3 subculture, well-grown resistance Multiple Buds cut and are transferred on root media Culture obtains Kan resistance and regenerates sweet wormwood plant to taking root. Preferably, in step S4, the method for the PCR detection are as follows: separately design synthesis expression cassette p35s-AaSPL2-nos The specific primer of AaSPL2 gene carries out PCR amplification in sequence, observes purpose in the UV lamp after agarose gel electrophoresis The positive strain of band is transgene abrotanum plant；The specific primer includes upstream primer, downstream primer, particular sequence It is respectively as follows: PF:gagagactggtgatttcagcg, PR:ttaatttgaacaaaagtagtcaaaa. In step S5, the HPLC-ELSD measures content of artemisinin in sweet wormwood, method are as follows: chromatographic column C-18 reverse phase silicon Rubber column gel column, mobile phase are methanol: water, methanol: the volume ratio of water be 70:30,30 DEG C of column temperature, flow velocity 1.0mL/min, 10 μ of sample volume L, 40 DEG C of evaporative light scattering detector drift tube temperature, amplification coefficient (gain) is 7, nebulizer gas pressure 5bar. Agrobacterium tumefaciems used is the biomaterial that there is public offering in market in step S3 of the present invention, can be from more companies As Australian CAMBIA company buys. The present invention also provides sweet wormwood AaSPL2 genes to improve the application in content of artemisinin in sweet wormwood. This invention demonstrates that AaSPL2 gene is the important transcription factor of salicylic acid signal pathway and qinghaosu metabolic engineering Important target spot.The present invention has broken the limit of qinghaosu biosynthesis with AaSPL2 genetic transformation sweet wormwood using genetic engineering means Fast bottleneck obtains the sweet wormwood plant of qinghaosu high yield, establishes the method stablized and improve content of artemisinin in sweet wormwood, is scale Production qinghaosu provides a new way. Beneficial effects of the present invention: The method provided by the invention for improving content of artemisinin in sweet wormwood using AaSPL2 gene, using genetic engineering side Key gene AaSPL2 is imported in sweet wormwood plant, obtains the transgene abrotanum strain that artemislnin content significantly improves by method, The content for turning AaSPL2 gene Artemisinin in Artemisia annuna can reach 20.6mg/g DW, be non-transformed common sweet wormwood (10.9mg/g DW) 1.8 times, content of artemisinin in sweet wormwood is significantly improved, provides high yield, stable new drug for the large-scale production of qinghaosu Source is of great significance. Detailed description of the invention Fig. 1 is AaSPL2 gene in the embodiment of the present invention 1 by MeJA induced map. Fig. 2 be the embodiment of the present invention 1 in AaSPL2 gene different tissues express spectra. Fig. 3 is 3 plant expression vector of the embodiment of the present invention: the building signal of pCAMBIA2300::p35S-AaSPL2-nos Figure. Fig. 4 is the content detection result figure of 5 transgenic Artemisinin in Artemisia annuna of the embodiment of the present invention. Specific embodiment Elaborate below to the embodiment of the present invention: the present embodiment carries out under the premise of the technical scheme of the present invention Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation Example. In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc. Molecular cloning: described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) Condition, or according to the normal condition proposed by manufacturer. Embodiment 1 Hormone MeJA plays a significant role in induction secondary metabolite.MeJA can induce a variety of secondary metabolites Synthesis, such as danshinolic acid, qinghaosu in Radix Salviae Miltiorrhizae.SBP class transcription factor is a kind of important transcription factor, raw in regulation plant It plays a crucial role in long development, secondary metabolism.The research that SPL regulates and controls qinghaosu in sweet wormwood at present not yet has been reported that. 1.MeJA handles sweet wormwood It (uses by the sweet wormwood plant that the hormone MeJA of 100mM handles 30 days sizes and originates in Chongqing tenth of the twelve Earthly Branches sun artemislnin content Higher sweet wormwood kind), screen the SBP class transcription factor induced by MeJA.Respectively hormone MeJA handle 0h, 1h, 3h, 6h, 9h, 12h and sweet wormwood blade is taken for 24 hours.It is placed in liquid nitrogen, the extraction for RNA. 2. the extraction of sweet wormwood genome total serum IgE A small amount of sweet wormwood young leaflet tablet is taken to be ground with mortar rapidly after liquid nitrogen flash freezer, addition fills 1mL TRIzol In the 1.5mL Eppendorf pipe of (TRIzol Reagents, GIBCO BRL, USA), sufficiently after oscillation, place at room temperature 5min adds 200 μ L chloroforms, with forced oscillation 15sec, after being placed at room temperature for 2-3min, is centrifuged 15min in 4 DEG C, 12,000g；By supernatant Liquid (about 600 μ L) sucks in clean 1.5mL Eppendorf pipe, and isometric isopropanol is added, is mixed by inversion, room temperature decentralization After setting 10min, 10min is centrifuged in 4 DEG C, 12,000g；Supernatant is abandoned, 75% ethyl alcohol of 1mL is added to clean, after oscillation, in 4 DEG C, 7, 500g is centrifuged 5min；It is dissolved in after drying at room temperature 15-20min in appropriate (30-50 μ L) RNAase-free water；With denaturing formaldehyde glue Electroresis appraisal total serum IgE quality, then measures rna content on spectrophotometer. 3. screening the SBP class transcription factor induced by MeJA 0h, 1h, 3h, 6h, 9h, 12h are handled in hormone MeJA respectively and takes sweet wormwood blade for 24 hours, extract sweet wormwood according to step 2 Genome total serum IgE.The expression that SBP genoid is detected by Q-PCR generates thermal map file by MeV 4.9, referring to Fig. 1. As shown in Figure 1, AaSPL2 is induced obviously by MeJA in sweet wormwood SPL, after MeJA is handled sweet wormwood plant 1 hour, expression quantity It increases, between 3 hours to 24, expression quantity is also in raised trend, shows the induction of AaSPL2 response MeJA, and AaSPL2 may Participate in the regulation of qinghaosu. 4, sweet wormwood different tissues expression pattern analysis Sweet wormwood plant (identical as kind in step 1) different tissues position (COT, the cotyledon of 3 months sizes are taken respectively；ML, Mature leaf；Meristem, separate living tissue；YL, tender leaf；Bud, bud), RNA is extracted according to step 2 method.It is examined by Q-PCR The expression for surveying SBP genoid generates thermal map file by MeV 4.9, referring to fig. 2.Fig. 2 shows different groups in sweet wormwood (COT, cotyledon are knitted in position；ML, mature leaf；Meristem, separate living tissue；YL, tender leaf；Bud, bud), AaSPL2 is presented Different expressions.AaSPL2 expression quantity in colored and blade is higher, has class with artemisinin synthesis pathway key enzyme gene As express spectra, screening obtain AaSPL2 be by MeJA induce important transcription factor. Therefore, complex chart 1 and Fig. 2's as a result, show AaSPL2 may have regulation artemisinin synthesis potentiality. The clone of 2 sweet wormwood AaSPL2 gene of embodiment The sweet wormwood genome total serum IgE obtained is obtained into the first chain cDNA by reverse transcriptase XL (AMV) reverse transcription, according to institute The coded sequence (as shown in SEQ ID NO:1) of sweet wormwood AaSPL2 gene is stated, the upstream and downstream that design amplifies complete encoder block is drawn Object, AaSPL2-PF:atggagtggaattgggacaa, AaSPL2-PR:ttaatttgaacaaaagtagtcaaaa, and upper Restriction endonuclease sites (this can be depending on the carrier of selection) is introduced respectively in trip and downstream primer, is carried to construct expression Body. Using the first chain cDNA as template, it is sequenced after PCR amplification.Determined dna sequence is by Shanghai Ying Junsheng Object Technology Service Co., Ltd is completed using 3730 automatic sequencers.Sequencing result shows cloned sweet wormwood AaSPL2 gene Sequence is consistent with sequence shown in SEQ ID NO:1. The KOD reaction system of pcr amplification reaction is as shown in table 1. Table 1 Constituent Content 5×KOD buffer 5μL dNTP 5μL MgSO4 2μL PF(10μM) 1μL PR(10μM) 1μL Template DNA 1μL KOD enzyme 1μL dH2O up to 50μL PCR response procedures are as follows: The present embodiment obtains the important transcription factor AaSPL2 of salicylic acid signal pathway using gene clone method from sweet wormwood Gene, to provide an important key gene by turning AaSPL2 gene raising content of artemisinin in sweet wormwood. The building of the plant binary expression vector of 3 gene containing AaSPL2 of embodiment 1. the building of intermediate vector pCAMBIA2300::p35S-gus-nos It selects pBI121 (Clontech) and pCAMBIA2300 (Australian CAMBIA research institute) is primary element, structure Build binary plant expression vector pCAMBIA2300::p35S-gus-nos.Specifically, HindIII and EcoRI double digestion pBI121 With pCAMBIA2300 plasmid；Recycle the gus expression cassette and pCAMBIA2300 large fragment of pBI121；Connect recovery product, conversion Screening takes out plasmid enzyme restriction verifying, obtains binary plant expression vector pCAMBIA2300::p35S-gus-nos.Wherein, digestion is anti- Answer system as shown in table 2. Table 2 Constituent Content 10 × buffer of digestion 5μL Carrier 20μL Restriction endonuclease 1 1μL Interior enzyme 2 1μL dH2O up to 50μL 2. the building of plant expression vector pCAMBIA2300::p35S-AaSPL2-nos Using the pCAMBIA2300::p35S-gus-nos of building as expression vector, obtained with clone in embodiment 1 The gus gene of AaSPL2 gene replacement thereon, obtains plant expression vector pCAMBIA2300::p35S-AaSPL2-nos, building Schematic diagram is as shown in Figure 3.Specifically, BamHI/SacI double digestion pCAMBIA2300::p35S-gus-nos, recycling PCAMBIA2300::p35S-nos large fragment is arrived AaSPL2 gene swapping by seamless Cloning Kit (Nanjing Novi praises) PCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2- is formed on pCAMBIA2300::p35S-nos Nos, conversion, picking monoclonal extract plasmid and do PCR detection and digestion verification, obtain plant expression vector pCAMBIA2300:: p35S-AaSPL2-nos.Wherein, seamless cloning reaction system is as shown in table 3. Table 3 Constituent Content Gene PCR product 1μL Linearized vector 3μL Seamless clone Buffer 4μL Seamless clone enzyme 2μL dH2O up to 20μL Salicylic acid signal pathway important gene AaSPL2 is operatively connectable to expression regulation sequence, shape by the present embodiment At the plant expression vector of the gene containing AaSPL2, which can be used for improving sweet wormwood in sweet wormwood by metabolic engineering strategies The content of element. The Agrobacterium tumefaciens mediated AaSPL2 gene genetic of embodiment 4 converts sweet wormwood and obtains transgene abrotanum plant 1. the acquisition of the double base plant expression vector Agrobacterium tumefaciems engineering bacteria of gene containing AaSPL2 By the plant binary expression vector pCAMBIA2300::p35S-AaSPL2-nos of the gene containing AaSPL2 in embodiment 2 Being transferred to Agrobacterium tumefaciems, (such as EHA105 has the biomaterial of public offering for market, can purchase from Australian CAMBIA company , strain number is Gambar 1), and carry out PCR verifying.Wherein, plasmid be transferred to Agrobacterium method it is as follows: 1 μ L plasmid adds Enter into 50 μ L Agrobacterium competence, ice bath 30 minutes, freeze 5 minutes in liquid nitrogen, 37 DEG C heat shock 3 minutes, add LB culture Base activates 3 hours in 37 DEG C of shaking tables, coated plate, identification. The result shows that the binary expression vector of gene plant containing AaSPL2 is successfully building up in Agrobacterium tumefaciens strain. 2. Agrobacterium tumefaciens mediated AaSPL2 genetic transformation sweet wormwood 2.1. the preculture of explant 75% ethyl alcohol of seeds of southernwood impregnates 1min, then impregnates 20min with 20%NaClO, and aseptic water washing 3-4 times is used Sterile blotting paper blots surface moisture, is inoculated in MS (Murashige and Skoog, 1962) solid medium of no hormone In, 25 DEG C, 16h/8h (light/dark) illumination cultivation can be obtained sweet wormwood aseptic seedling.After seedling length to 5cm or so, clip Tests for sterility explant is for converting. 2.2. the co-cultivation of Agrobacterium and explant The blade explant is gone in co-culture medium (100 μm of ol/L of 1/2MS+AS), dropwise addition, which contains, to be activated The binary expression vector of gene plant containing AaSPL2 Agrobacterium tumefaciems engineering bacteria 1/2MS suspension, make explant and bacterium Liquid comes into full contact with, 28 DEG C of dark culture 3d.It is outstanding in the 1/2MS fluid nutrient medium of the Agrobacterium tumefaciems without target gene with dropwise addition The blade explant of liquid is control. 2.3. the screening of resistance regeneration plant The sweet wormwood explant for co-culturing 3d is transferred to germination screening and culturing medium (MS+6-BA 0.5mg/L+NAA 0.05mg/L+Kan 50mg/L+Cb 500mg/L) in 25 DEG C, 16h/8h illumination cultivation, squamous subculture is primary every two weeks, warp It can be obtained Kan resistance Multiple Buds after crossing 2-3 subculture.Well-grown resistance Multiple Buds are cut and are transferred to root media It is cultivated on (1/2MS+Cb 125mg/L) to taking root, to obtain Kan resistance regeneration sweet wormwood plant. 3. the PCR of transgene abrotanum plant is detected (sweet wormwood is extracted by DNA extraction kit (Tiangeng) by template of the Kan resistance of acquisition regeneration sweet wormwood plant DNA The DNA of plant), forward direction is separately designed according to expression cassette p35s-AaSPL2-nos sequence p35s and AaSPL2 where target gene Design of primers and reverse primer detect target gene, detection primer PF:gagagactggtgatttcagcg, PR: ttaatttgaacaaaagtagtcaaaa.Wherein, it using DNA as template, is expanded by TAKARA enzyme (Dalian treasured bioengineering), Rtaq-MIX enzyme reaction system is as shown in table 4 and program is as follows: Table 4 Constituent Content 2×rtaq-MIX buffer 5μL PF(10μM) 1μL PR(10μM) 1μL Template DNA 1μL Rtaq enzyme 1μL dH2O up to 20μL PCR response procedures are as follows: The result shows that the specific DNA fragment of 1000bp, i.e. agarose can be amplified using designed PCR special primer Purpose band is observed after gel electrophoresis in the UV lamp, the Kan resistance regeneration sweet wormwood plant of acquisition is to turn AaSPL2 gene Sweet wormwood plant.And when using non-transformed sweet wormwood genomic DNA as template, do not amplify any segment. The plant expression vector is converted Agrobacterium tumefaciems by the present embodiment, obtain for convert sweet wormwood containing AaSPL2 The Agrobacterium tumefaciens strain of gene plant expression vector converts sweet wormwood using constructed Agrobacterium tumefaciens strain, obtains through PCR The transgene abrotanum plant of detection.The acquisition of transgene abrotanum plant is that the sweet wormwood strain of the higher artemislnin content of screening acquisition mentions Direct material is supplied. Embodiment 5 utilizes artemislnin content in HPLC-ELSD measurement transgene abrotanum The preparation of 1.HPLC-ELSD condition and system suitability and standard solution HPLC: using 2695 system of water alliance, and chromatographic column is C-18 reverse phase silica gel column (SymmetryShieldTM C18,5 μm, 250 × 4.6mm, Waters), mobile phase is methanol: water, methanol: the volume ratio of water For 70:30,30 DEG C of column temperature, flow velocity 1.0mL/min, 10 μ L of sample volume, sensitivity (AUFS=1.0), theoretical cam curve presses sweet wormwood Plain peak, which calculates, is not less than 2000. ELSD: 2420 system of water alliance is used, 40 DEG C of evaporative light scattering detector drift tube temperature, is amplified Coefficient (gain) is 7, nebulizer gas pressure 5bar. Precision weighs qinghaosu standard items (Sigma company) 2.0mg and is completely dissolved with 1mL methanol, obtains 2mg/mL sweet wormwood Plain standard solution, be stored in -20 DEG C it is spare. Mobile phase is methanol (methanol): water in the present invention, when ratio is 70%:30%, the retention time of qinghaosu For 5.1min, peak type is good.Theoretical cam curve is calculated by qinghaosu is not less than 2000. 2. the production of standard curve Above-mentioned standard product solution is distinguished to sample introduction 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l under corresponding chromatographic condition and records map And chromatographic parameter, regression analysis is carried out to standard items content (X, μ g) with peak area (Y) respectively.It is green in the present invention by research Good log-log linear relationship is presented in artemisin in 4-20 μ g range.The log-log equation of linear regression of Qinghaosu Are as follows: Y=1.28e+000X+4.71e+000, R=0.979546. 3. the preparation of sample and the measurement of artemislnin content The extraction process of qinghaosu is based on the method reported in Van Nieuwerburgh et al. (2006): taking a small amount of Fresh sweet wormwood blade (1-2g fresh weight), is immersed into 10ml chloroform in 50ml test tube and sways 1 minute, leachate is fallen Enter to make in new test tube chloroform volatilization completely, 3ml dehydrated alcohol is taken sufficiently to dissolve extract, is detected for HPLC.Meanwhile chloroform Blade collection after extraction, which is put into 60 degree of baking ovens, is dried, and weighing (calculates the dry weight of sweet wormwood blade)； Artemislnin content is measured using HPLC-ELSD, sample feeding volume is 20 μ l, substitutes into linear regression according to peak area Equation calculation goes out the artemislnin content (mg) in sample, then divided by the sweet wormwood leaf dry weight (g) of sample, to calculate sweet wormwood plant The content of middle qinghaosu. After measured, the content of transfer AaSPL2 gene Artemisinin in Artemisia annuna of the present invention can reach 20.6mg/g DW, be 1.8 times (as shown in Figure 4) of non-transformed common sweet wormwood (10.9mg/g DW), AaSPL2 gene significantly improve sweet wormwood in sweet wormwood Cellulose content. The present embodiment determines artemislnin content in transgene abrotanum using HPLC-ELSD method, using conversion AaSPL2 base The metabolic engineering strategies of cause obtain the sweet wormwood plant of qinghaosu high yield, provide a kind of ideal side for large-scale production qinghaosu Method. Sequence table <110>Second Military Medical University, PLA <120>a kind of method for improving content of artemisinin in sweet wormwood using AaSPL2 gene <130>specification, claims <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 954 <212> DNA <213>sweet wormwood (Artemisia annua) <400> 1 atggagtgga attgggacaa taatgaagtt cccaagagtt tagcggtttc aagccacgaa 60 aatggcgagt atttgggggg tgaagatgtg caaaggagtt tttctaatga tataattgag 120 gaaggctcgg ttatatctgg tgaagcacta tttggtttga agctcgggca agagacatat 180 aatcaagata atcttagaat gagctcattc ccattagtac ctacctcttt gagttctccg 240 gttgtgaaaa aatctagagc ggtgtaccaa agttcacatt cgccacgttg tcaagttgag 300 gggtgcaacc ttgaccttgt gtcggctaaa gattaccacc gtcggcataa aatttgtgct 360 gatcattcta agagcccaaa agttgttgtt ggggggatgg aacgaaggtt ttgtcagcaa 420 tgtagcaggt tgcatgattt atcggagttt gacgatagga agcgtagttg tcgcaggcgc 480 ctctcggcac acaatgctag acgtcgtagg ccacaatcag aggacaagtt cagccccaca 540 catcgaagac catatatggg tttctttgtg aacggggtct cgacctcgac cccatcacca 600 aattcgacac cacaaagttc atctaacttt aaacgtgaag atgttgacat gatctcgagt 660 ggtgccttat cggacatctc gtcgtctttc catggggtca cacctagact tgctaaccac 720 gagggtttag atgcaaactc taactcgatc tgtaagatgg aagttcgaca ttctttctct 780 cttcaagcca cgagctcttg gggattcaac agtcgcgatg agccctcttc ctttgatcaa 840 ttcattaatg gacacaacgc cggcttgacg caacatggga caccactcga gatgcaacat 900 acccgaaaca ctcaaattcc acctcatgat tttgactact tttgttcaaa ttaa 954 Claims (10) ] 1. a kind of method for improving content of artemisinin in sweet wormwood using AaSPL2 gene, includes the following steps: S1: sweet wormwood key gene AaSPL2 is obtained using gene clone method； AaSPL2 gene: being operatively connectable to expression regulation sequence by S2, and the plant for forming the gene containing AaSPL2, which is expressed, to be carried Body； S3: converting Agrobacterium tumefaciems for the plant expression vector of the gene containing AaSPL2, obtain for convert sweet wormwood containing AaSPL2 The Agrobacterium tumefaciens strain of gene plant expression vector； S4: converting sweet wormwood using constructed Agrobacterium tumefaciens strain, obtains the transgene abrotanum through PCR test positive and plants Strain； S5: HPLC-ELSD measurement is carried out to artemislnin content in the transgene abrotanum of acquisition, it is significant that screening obtains artemislnin content The transgene abrotanum plant of raising. 2. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that Step S1 includes the following steps: to extract sweet wormwood genome total serum IgE；Sweet wormwood genome total serum IgE is obtained into the first chain by reverse transcription cDNA；It using the first chain cDNA as template, is sequenced after PCR amplification, obtains and correct sweet wormwood AaSPL2 gene is sequenced； The DNA sequence dna according to shown in SEQ ID NO.1 when PCR amplification, design amplifies the upstream and downstream primer of complete encoder block, described The nucleotide sequence of upstream and downstream primer is respectively as follows: AaSPL2-PF:atggagtggaattgggacaa；AaSPL2-PR: ttaatttgaacaaaagtagtcaaaa。 3. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that In step S2, it is described building the gene containing AaSPL2 plant expression vector include the following steps: select pBI121 and PCAMBIA2300 is primary element, constructs binary plant expression vector pCAMBIA2300::p35S-gus-nos；BamHI is used again With SacI double digestion carrier pCAMBIA2300::p35S-gus-nos, pCAMBIA2300::p35S-nos large fragment is recycled, it will PCAMBIA2300::p35S-pCAMBIA2300: is formed in AaSPL2 gene swapping to pCAMBIA2300::p35S-nos: P35S-gus-AaSPL2-nos, conversion, picking monoclonal extract plasmid and do PCR detection and digestion verification. 4. the method according to claim 3 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that The building binary plant expression vector pCAMBIA2300::p35S-gus-nos include the following steps: with HindIII and EcoRI double digestion pBI121 and pCAMBIA2300 plasmid；Recycle the gus expression cassette and pCAMBIA2300 large fragment of pBI121； Recovery product is connected, transformation and selection takes out plasmid enzyme restriction verifying. 5. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that It is described to convert the preculture for including the following steps: explant in step S4；The co-cultivation of Agrobacterium and explant；Resistance regeneration The screening of plant. 6. the method according to claim 5 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that The preculture of the explant includes the following steps: that seeds of southernwood impregnates 1min with 75% ethyl alcohol, then is impregnated with 20%NaClO 20min, aseptic water washing 3-4 times blot surface moisture with sterile blotting paper, are inoculated in the MS solid medium of no hormone, Illumination cultivation 16h, dark culture 8h, can be obtained sweet wormwood aseptic seedling at 25 DEG C, after seedling length to 5~8cm, clip tests for sterility Explant is for converting. 7. the method according to claim 5 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that The co-cultivation of the Agrobacterium and explant includes the following steps: the tests for sterility explant going to co-culture medium In, it is added dropwise containing the activated binary expression vector of gene plant containing the AaSPL2 pCAMBIA2300::p35S- The 1/2MS suspension of the Agrobacterium tumefaciems engineering bacteria of pCAMBIA2300::p35S-gus-AaSPL2-nos, makes explant and bacterium solution It comes into full contact with, 28 DEG C of dark culture 3d. 8. the method according to claim 5 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that The screening of the resistance regeneration plant includes the following steps: that the sweet wormwood explant by the co-cultivation 3d is transferred to germination screening On culture medium at 25 DEG C illumination cultivation 16h, dark culture 8h, squamous subculture is primary every two weeks, can obtain after 2-3 subculture Kan resistance Multiple Buds are obtained, well-grown resistance Multiple Buds are cut to be transferred on root media and are cultivated to taking root, Kan is obtained Resistance regenerates sweet wormwood plant. 9. the method according to claim 1 for improving content of artemisinin in sweet wormwood using AaSPL2 gene, which is characterized in that In step S4, the method for the PCR detection are as follows: separately design AaSPL2 gene in expression cassette p35s-AaSPL2-nos sequence Specific primer carries out PCR amplification, observes that the positive strain of purpose band is after agarose gel electrophoresis in the UV lamp Transgene abrotanum plant；The specific primer includes upstream primer, downstream primer, and particular sequence is respectively as follows: PF: Gagagactggtgatttcagcg, PR:ttaatttgaacaaaagtagtcaaaa. 10. sweet wormwood AaSPL2 gene is improving the application in content of artemisinin in sweet wormwood. https://patents.google.com/patent/CN110438148A/en 一种利用AaSPL2基因提高青蒿中青蒿素含量的方法 技术领域 本发明属于生物技术领域。具体涉及的是一种提高青蒿素含量的方法，特别涉及是一种利用AaSPL2基因提高青蒿中青蒿素含量的方法。 背景技术 青蒿(Artemisia annua L.)是菊科蒿属的一年生草本植物。青蒿素(artemisinin)是从其地上部分分离的一种含有过氧桥结构的倍半萜内酯化合物，是目前世界上公认的最有效的治疗疟疾的药物，特别是对于脑型疟疾和抗氯喹疟疾具有速效和低毒的特点。目前，世界卫生组织推荐的最有效的治疗疟疾的方法就是青蒿素联合疗法(ACTs)。另外，随着对青蒿素药理研究的逐步深入，科学家发现青蒿素及其衍生物还具有抗炎、抗血吸虫、抗肿瘤以及免疫调节的功能。可见青蒿素是一种极具潜力的天然药物。 目前青蒿素的主要来源是从青蒿植株的地上部分提取，然而青蒿中青蒿素的含量非常低(0.01％-1％)，使得这种药物的大规模商业化生产受到了限制。由于青蒿素结构复杂，人工合成难度大，产量低，成本高，不具有可行性。有人尝试用组织培养和细胞工程的方法来生产青蒿素，然而青蒿素在愈伤组织中含量低于干重的0.1％，在芽中最高也只有干重的0.16％，而大多数研究在根中没有检测到青蒿素。因此利用组织培养及细胞工程来生产青蒿素的可行性也不高。 经对现有技术文献检索发现，Qian Shen等在《New Phytologist》(《新植物学家》，2016年210期1269-1281页)发表了题为“The jasmonate-responsiveAaMYC2transcription factor positively regulates artemisinin biosynthesis inArtemisia annua”(“受甲基茉莉酸响应的AaMYC2转录因子正调控青蒿中青蒿素的生物合成”)的论文，报道通过在青蒿中过表达受激素甲基茉莉酸响应的AaMYC2，使青蒿素含量提高了1.6倍。因此，通过过表达受甲基茉莉酸响应的基因为提高青蒿中青蒿素的含量提供了一条可行的方法。 发明内容 本发明的目的在于提供一种利用AaSPL2基因提高青蒿中青蒿素含量的方法，将青蒿中克隆出的AaSPL2基因转入青蒿中得到转基因AaSPL2青蒿，该转基因AaSPL2青蒿中青蒿素含量显著提高，从而实现利用青蒿大规模生产青蒿素。 本发明是通过以下技术方案实现的：本发明从青蒿中克隆AaSPL2基因，构建含AaSPL2基因的植物表达载体，用根癌农杆菌介导，将AaSPL2基因导入青蒿并再生出植株；PCR检测外源目的基因AaSPL2的整合情况，高效液相色谱-蒸发光散射检测器(HPLC-ELSD)测定青蒿中青蒿素含量，筛选获得青蒿素含量提高的转基因青蒿植株。 本发明提供的利用AaSPL2基因提高青蒿中青蒿素含量的方法，包括如下步骤： S1：采用基因克隆方法获得青蒿关键酶基因AaSPL2； S2：把AaSPL2基因可操作性地连接于表达调控序列，构建含AaSPL2基因的植物表达载体； S3：将含AaSPL2基因的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含AaSPL2基因植物表达载体的根癌农杆菌菌株； S4：利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测为阳性的转基因青蒿植株； S5：对获得的转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量显著提高的转基因青蒿植株。 优选的，步骤S1：提取青蒿基因组总RNA；将青蒿基因组总RNA通过反转录获得第一链cDNA；根据如SEQ ID NO.1所示的青蒿AaSPL2基因的编码序列设计扩增出完整编码框的上下游引物，并在上游和下游引物上分别引入限制性内切酶位点，以便构建表达载体；以所述第一链cDNA为模板，经PCR扩增后进行测序，获得测序正确的青蒿AaSPL2基因，其中PCR扩增所用的上、下游引物的核苷酸序列分别为：AaSPL2-PF：atggagtggaattgggacaa，AaSPL2-PR：ttaatttgaacaaaagtagtcaaaa。 优选的，步骤S2中，所述构建含AaSPL2基因的植物表达载体包括如下步骤：选用pBI121和pCAMBIA2300为基本元件，构建双元植物表达载体pCAMBIA2300::p35S-gus-nos；再用BamHI和SacI双酶切载体pCAMBIA2300::p35S-gus-nos，回收pCAMBIA2300::p35S-nos大片段，通过无缝克隆试剂盒将AaSPL2基因交换到pCAMBIA2300::p35S-nos上形成pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos，转化，挑取单克隆，提取质粒做PCR检测和酶切验证。 更优选的，所述构建双元植物表达载体pCAMBIA2300::p35S-gus-nos包括如下步骤：用HindIII和EcoRI双酶切pBI121和pCAMBIA2300质粒；回收pBI121的gus表达盒和pCAMBIA2300大片段；连接回收产物，转化筛选，抽质粒酶切验证。 优选的，步骤S4中，所述转化包括如下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。 更优选的，所述外植体的预培养包括如下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃下光照培养16h、暗培养8h，即可获得青蒿无菌苗，待苗长至5～8cm后，剪取无菌苗叶片外植体用于转化。 更优选的，所述农杆菌与外植体的共培养包括如下步骤：将所述无菌苗叶片外植体转到共培养培养基中，滴加含活化好的所述含AaSPL2基因植物双元表达载体pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3d。 更优选的，所述抗性再生植株的筛选包括如下步骤：将所述的共培养3d的青蒿外植体转入到发芽筛选培养基上于25℃下光照培养16h、暗培养8h，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，获得Kan抗性再生青蒿植株。 优选的，步骤S4中，所述PCR检测的方法为：分别设计合成表达盒p35s-AaSPL2-nos序列上AaSPL2基因的特异性引物进行PCR扩增，琼脂糖凝胶电泳后在紫外灯下观察到目的条带的阳性株系即为转基因青蒿植株；所述特异性引物包括上游引物、下游引物，具体序列分别为：PF：gagagactggtgatttcagcg，PR：ttaatttgaacaaaagtagtcaaaa。 步骤S5中，所述的HPLC-ELSD测定青蒿中青蒿素含量，方法为：色谱柱C-18反相硅胶柱，流动相为甲醇：水，甲醇:水的体积比为70：30，柱温30℃，流速1.0mL/min，进样量10μL，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar。 本发明步骤S3中所用根癌农杆菌为市场有公开出售的生物材料，可以从多家公司如澳大利亚CAMBIA公司购得。 本发明还提供青蒿AaSPL2基因在提高青蒿中青蒿素含量中的应用。 本发明证明：AaSPL2基因是水杨酸信号途径重要的转录因子及青蒿素代谢工程的重要靶点。本发明采用基因工程手段，用AaSPL2基因转化青蒿，打破了青蒿素生物合成的限速瓶颈，获得青蒿素高产的青蒿植株，建立了稳定提高青蒿中青蒿素含量的方法，为规模化生产青蒿素提供了一条新途径。 本发明的有益效果： 本发明提供的利用AaSPL2基因提高青蒿中青蒿素含量的方法，采用基因工程方法，将关键酶基因AaSPL2导入青蒿植株中，获得了青蒿素含量显著提高的转基因青蒿株系，转AaSPL2基因青蒿中青蒿素的含量最高可达到20.6mg/g DW，是非转化普通青蒿(10.9mg/gDW)的1.8倍，显著提高了青蒿中青蒿素含量，为青蒿素的规模化生产提供高产、稳定的新药源，具有重要意义。 附图说明 图1为本发明实施例1中AaSPL2基因受MeJA诱导图。 图2为本发明实施例1中AaSPL2基因在不同组织的表达谱。 图3为本发明实施例3植物表达载体：pCAMBIA2300::p35S-AaSPL2-nos的构建示意图。 图4为本发明实施例5中转基因青蒿中青蒿素的含量检测结果图。 具体实施方式 下面对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。 下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。 实施例1 激素MeJA在诱导次生代谢产物中具有重要作用。MeJA能诱导多种次生代谢产物的合成，如丹参中的丹酚酸、青蒿素等。SBP类转录因子是一类重要的转录因子，在调控植物生长发育、次生代谢中起关键作用。目前青蒿中SPL调控青蒿素的研究尚未有报道。 1.MeJA处理青蒿 通过100mM的激素MeJA处理30天大小的青蒿植株(采用产于重庆酉阳青蒿素含量较高的青蒿品种)，筛选受MeJA诱导的SBP类转录因子。分别在激素MeJA处理0h、1h、3h、6h、9h、12h和24h取青蒿叶片。置于液氮中，用于RNA的提取。 2.青蒿基因组总RNA的提取 取少量青蒿幼嫩叶片，用液氮速冻后，迅速用研钵研碎，加入盛有1mL TRIzol(TRIzol Reagents,GIBCO BRL,USA)的1.5mL Eppendorf管中，充分振荡后，于室温下放置5min，加200μL氯仿，用力振荡15sec，室温放置2-3min后，于4℃、12,000g离心15min；将上清液(约600μL)吸入干净的1.5mL Eppendorf管中，加入等体积的异丙醇，颠倒混匀，室温下放置10min后，于4℃、12,000g离心10min；弃上清，加1mL 75％乙醇清洗，振荡后，于4℃、7,500g离心5min；室温干燥15-20min后溶于适量(30-50μL)RNAase-free水中；用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。 3.筛选受MeJA诱导的SBP类转录因子 分别在激素MeJA处理0h、1h、3h、6h、9h、12h和24h取青蒿叶片，按照步骤2提取青蒿基因组总RNA。通过Q-PCR检测SBP类基因的表达水平，通过MeV 4.9生成热图文件，参见图1。由图1可知，在青蒿SPL中AaSPL2受MeJA诱导明显，在MeJA处理青蒿植株1小时后，其表达量升高，在3小时到24之间，表达量也呈升高的趋势，表明AaSPL2响应MeJA的诱导，AaSPL2可能参与青蒿素的调控。 4、青蒿不同组织表达谱分析 分别取3个月大小的青蒿植株(与步骤1中品种相同)不同组织部位(COT，子叶；ML，成熟叶片；Meristem，分生组织；YL，嫩叶；Bud，花蕾)，按照步骤2方法提取RNA。通过Q-PCR检测SBP类基因的表达水平，通过MeV 4.9生成热图文件，参见图2。图2表明，在青蒿的不同组织部位中(COT，子叶；ML，成熟叶片；Meristem，分生组织；YL，嫩叶；Bud，花蕾)，AaSPL2呈现不同的表达水平。AaSPL2在花和叶片中表达量较高，与青蒿素合成途径关键酶基因具有类似的表达谱，筛选得到AaSPL2是受MeJA诱导的重要转录因子。 因此，综合图1和图2的结果，表明AaSPL2可能具有调控青蒿素合成的潜力。 实施例2青蒿AaSPL2基因的克隆 将所获的青蒿基因组总RNA通过反转录酶XL(AMV)反转录获得第一链cDNA，根据所述青蒿AaSPL2基因的编码序列(如SEQ ID NO:1所示)，设计扩增出完整编码框的上下游引物，AaSPL2-PF:atggagtggaattgggacaa，AaSPL2-PR:ttaatttgaacaaaagtagtcaaaa，并在上游和下游引物上分别引入限制性内切酶位点(这可视选用的载体而定)，以便构建表达载体。 以所述的第一链cDNA为模板，经PCR扩增后进行测序。DNA序列测定由上海英骏生物技术服务有限公司采用3730自动测序仪完成。测序结果表明，所克隆的青蒿AaSPL2基因序列与SEQ ID NO:1所示的序列一致。 PCR扩增反应的KOD反应体系如表1所示。 表1 组成成分 含量 5×KOD buffer 5μL dNTP 5μL MgSO₄ 2μL PF(10μM) 1μL PR(10μM) 1μL 模板DNA 1μL KOD酶 1μL dH₂O up to 50μL PCR反应程序如下： 本实施例采用基因克隆方法从青蒿中获得水杨酸信号途径重要转录因子AaSPL2基因，为通过转AaSPL2基因提高青蒿中青蒿素含量提供了一个重要关键酶基因。 实施例3含AaSPL2基因的植物双元表达载体的构建 1.中间载体pCAMBIA2300::p35S-gus-nos的构建 选用pBI121(Clontech)和pCAMBIA2300(澳大利亚CAMBIA研究所)为基本元件，构建双元植物表达载体pCAMBIA2300::p35S-gus-nos。具体地，HindIII和EcoRI双酶切pBI121和pCAMBIA2300质粒；回收pBI121的gus表达盒和pCAMBIA2300大片段；连接回收产物，转化筛选，抽质粒酶切验证，得到双元植物表达载体pCAMBIA2300::p35S-gus-nos。其中，酶切反应体系如表2所示。 表2 组成成分 含量 酶切10×buffer 5μL 载体 20μL 内切酶1 1μL 内酶2 1μL dH2O up to 50μL 2.植物表达载体pCAMBIA2300::p35S-AaSPL2-nos的构建 以构建的pCAMBIA2300::p35S-gus-nos为表达载体，用实施例1中克隆得到的AaSPL2基因替换其上的gus基因，得到植物表达载体pCAMBIA2300::p35S-AaSPL2-nos，构建示意图如图3所示。具体地，BamHI/SacI双酶切pCAMBIA2300::p35S-gus-nos，回收pCAMBIA2300::p35S-nos大片段，通过无缝克隆试剂盒(南京诺维赞)将AaSPL2基因交换到pCAMBIA2300::p35S-nos上形成pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos，转化，挑取单克隆，提取质粒做PCR检测和酶切验证，得到植物表达载体pCAMBIA2300::p35S-AaSPL2-nos。其中，无缝克隆反应体系如表3所示。 表3 组成成分 含量 基因PCR产物 1μL 线性化载体 3μL 无缝克隆Buffer 4μL 无缝克隆酶 2μL dH₂O up to 20μL 本实施例将水杨酸信号途径重要基因AaSPL2可操作性地连接于表达调控序列，形成含AaSPL2基因的植物表达载体，该表达载体可用于通过代谢工程策略来提高青蒿中青蒿素的含量。 实施例4根癌农杆菌介导AaSPL2基因遗传转化青蒿获得转基因青蒿植株 1.含AaSPL2基因双元植物表达载体根癌农杆菌工程菌的获得 将实施例2中含AaSPL2基因的植物双元表达载体pCAMBIA2300::p35S-AaSPL2-nos转入根癌农杆菌(如EHA105，为市场有公开出售的生物材料，可以从澳大利亚CAMBIA公司购得，菌株编号为Gambar 1)，并进行PCR验证。其中，质粒转入农杆菌的方法如下：1μL质粒加入到50μL农杆菌感受态中，冰浴30分钟，液氮中冷冻5分钟，37℃热激3分钟，再加入LB培养基在37℃摇床活化3小时，涂板，鉴定。 结果表明，含AaSPL2基因植物双元表达载体已成功构建到根癌农杆菌菌株中。 2.根癌农杆菌介导AaSPL2基因转化青蒿 2.1.外植体的预培养 青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS(Murashige and Skoog,1962)固体培养基中，25℃、16h/8h(light/dark)光照培养，即可获得青蒿无菌苗。待苗长至5cm左右后，剪取无菌苗叶片外植体用于转化。 2.2.农杆菌与外植体的共培养 将所述的叶片外植体转到共培养培养基(1/2MS+AS 100μmol/L)中，滴加含活化好的所述含AaSPL2基因植物双元表达载体的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3d。以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。 2.3.抗性再生植株的筛选 将共培养3d的青蒿外植体转入到发芽筛选培养基(MS+6-BA 0.5mg/L+NAA0.05mg/L+Kan 50mg/L+Cb 500mg/L)上于25℃、16h/8h光照培养，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽。将生长良好的抗性丛生芽剪下转入生根培养基(1/2MS+Cb 125mg/L)上培养至生根，从而获得Kan抗性再生青蒿植株。 3.转基因青蒿植株的PCR检测 以获得的Kan抗性再生青蒿植株DNA为模板(通过DNA提取试剂盒(天根)提取青蒿植株的DNA)，根据目的基因所在表达盒p35s-AaSPL2-nos序列p35s和AaSPL2分别设计正向引物设计和反向引物对目的基因进行检测，检测引物为PF：gagagactggtgatttcagcg，PR：ttaatttgaacaaaagtagtcaaaa。其中，以DNA为模板，通过TAKARA酶(大连宝生物工程)扩增，rtaq-MIX酶反应体系如表4所示及程序如下： 表4 组成成分 含量 2×rtaq-MIX buffer 5μL PF(10μM) 1μL PR(10μM) 1μL 模板DNA 1μL rtaq酶 1μL dH₂O up to 20μL PCR反应程序如下： 结果表明，利用所设计的PCR特异引物，能扩增出1000bp的特异DNA片段，即琼脂糖凝胶电泳后在紫外灯下观察到目的条带的，获得的Kan抗性再生青蒿植株为转AaSPL2基因青蒿植株。而以非转化青蒿基因组DNA为模板时，没有扩增出任何片段。 本实施例将所述的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含AaSPL2基因植物表达载体的根癌农杆菌菌株，利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测的转基因青蒿植株。转基因青蒿植株的获得为筛选获得较高青蒿素含量的青蒿株系提供了直接素材。 实施例5利用HPLC-ELSD测定转基因青蒿中青蒿素含量 1.HPLC-ELSD条件及系统适用性以及标准溶液的配制 HPLC：采用water alliance 2695系统，色谱柱为C-18反相硅胶柱(SymmetryShieldTM C18，5μm，250×4.6mm，Waters)，流动相为甲醇：水，甲醇：水的体积比为70：30，柱温30℃，流速1.0mL/min，进样量10μL，灵敏度(AUFS＝1.0)，理论塔板数按青蒿素峰计算不低于2000。 ELSD：采用water alliance 2420系统，蒸发光散射检测器漂移管温度40℃，放大系数(gain)为7，载气压力5bar。 精密称取青蒿素标准品(Sigma公司)2.0mg用1mL甲醇完全溶解，得到2mg/mL青蒿素标准品溶液，保存于-20℃备用。 本发明中流动相为甲醇(methanol)：水，比例为70％：30％时，青蒿素的保留时间为5.1min，峰型良好。理论塔板数按青蒿素计算不低于2000。 2.标准曲线的制作 将上述标准品溶液在相应色谱条件下分别进样2μl，4μl，6μl，8μl，10μl记录图谱及色谱参数，分别以峰面积(Y)对标准品含量(X,μg)进行回归分析。通过研究，本发明中青蒿素在4-20μg范围内呈现良好的log-log线性关系。青蒿素对照品的log-log线性回归方程为：Y＝1.28e+000X+4.71e+000，R＝0.979546。 3.样品的制备和青蒿素含量的测定 青蒿素的提取过程基于Van Nieuwerburgh et al.(2006)中报道的方法：取少量新鲜的青蒿叶片(1-2g鲜重)，于50ml试管中将其浸没在10ml氯仿中摇荡1分钟，将浸出液倒入新的试管中使氯仿挥发完全，取3ml无水乙醇充分溶解提取物，用于HPLC检测。同时，氯仿提取后的叶片收集放入60度烘箱进行烘干，称重(计算青蒿叶片的干重)； 采用HPLC-ELSD测定青蒿素含量，样品进样体积为20μl，根据峰面积代入线形回归方程计算出样品中的青蒿素含量(mg)，再除以样品的青蒿叶干重(g)，从而计算出青蒿植株中青蒿素的含量。 经测定，本发明中转AaSPL2基因青蒿中青蒿素的含量最高可达到20.6mg/g DW，是非转化普通青蒿(10.9mg/g DW)的1.8倍(如图4所示)，AaSPL2基因显著提高了青蒿中青蒿素含量。 本实施例采用HPLC-ELSD法测定了转基因青蒿中青蒿素含量，采用转化AaSPL2基因的代谢工程策略获得了青蒿素高产的青蒿植株，为规模化生产青蒿素提供了一种理想方法。 序列表 <110> 中国人民解放军第二军医大学 <120> 一种利用AaSPL2基因提高青蒿中青蒿素含量的方法 <130> 说明书，权利要求书 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 954 <212> DNA <213> 青蒿(Artemisia annua) <400> 1 atggagtgga attgggacaa taatgaagtt cccaagagtt tagcggtttc aagccacgaa 60 aatggcgagt atttgggggg tgaagatgtg caaaggagtt tttctaatga tataattgag 120 gaaggctcgg ttatatctgg tgaagcacta tttggtttga agctcgggca agagacatat 180 aatcaagata atcttagaat gagctcattc ccattagtac ctacctcttt gagttctccg 240 gttgtgaaaa aatctagagc ggtgtaccaa agttcacatt cgccacgttg tcaagttgag 300 gggtgcaacc ttgaccttgt gtcggctaaa gattaccacc gtcggcataa aatttgtgct 360 gatcattcta agagcccaaa agttgttgtt ggggggatgg aacgaaggtt ttgtcagcaa 420 tgtagcaggt tgcatgattt atcggagttt gacgatagga agcgtagttg tcgcaggcgc 480 ctctcggcac acaatgctag acgtcgtagg ccacaatcag aggacaagtt cagccccaca 540 catcgaagac catatatggg tttctttgtg aacggggtct cgacctcgac cccatcacca 600 aattcgacac cacaaagttc atctaacttt aaacgtgaag atgttgacat gatctcgagt 660 ggtgccttat cggacatctc gtcgtctttc catggggtca cacctagact tgctaaccac 720 gagggtttag atgcaaactc taactcgatc tgtaagatgg aagttcgaca ttctttctct 780 cttcaagcca cgagctcttg gggattcaac agtcgcgatg agccctcttc ctttgatcaa 840 ttcattaatg gacacaacgc cggcttgacg caacatggga caccactcga gatgcaacat 900 acccgaaaca ctcaaattcc acctcatgat tttgactact tttgttcaaa ttaa 954 Claims (10) 1.一种利用AaSPL2基因提高青蒿中青蒿素含量的方法，包括如下步骤： S1：采用基因克隆方法获得青蒿关键酶基因AaSPL2； S2：把AaSPL2基因可操作性地连接于表达调控序列，形成含AaSPL2基因的植物表达载体； S3：将含AaSPL2基因的植物表达载体转化根癌农杆菌，获得用于转化青蒿的含AaSPL2基因植物表达载体的根癌农杆菌菌株； S4：利用所构建的根癌农杆菌菌株转化青蒿，获得经PCR检测为阳性的转基因青蒿植株； S5：对获得的转基因青蒿中青蒿素含量进行HPLC-ELSD测定，筛选获得青蒿素含量显著提高的转基因青蒿植株。 2.根据权利要求1所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，步骤S1包括如下步骤：提取青蒿基因组总RNA；将青蒿基因组总RNA通过反转录获得第一链cDNA；以所述第一链cDNA为模板，经PCR扩增后进行测序，获得测序正确的青蒿AaSPL2基因；PCR扩增时根据SEQ ID NO.1所示的DNA序列，设计扩增出完整编码框的上、下游引物，所述上、下游引物的核苷酸序列分别为：AaSPL2-PF:atggagtggaattgggacaa；AaSPL2-PR:ttaatttgaacaaaagtagtcaaaa。 3.根据权利要求1所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，步骤S2中，所述构建含AaSPL2基因的植物表达载体包括如下步骤：选用pBI121和pCAMBIA2300为基本元件，构建双元植物表达载体pCAMBIA2300::p35S-gus-nos；再用BamHI和SacI双酶切载体pCAMBIA2300::p35S-gus-nos，回收pCAMBIA2300::p35S-nos大片段，将AaSPL2基因交换到pCAMBIA2300::p35S-nos上形成pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos，转化，挑取单克隆，提取质粒做PCR检测和酶切验证。 4.根据权利要求3所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，所述构建双元植物表达载体pCAMBIA2300::p35S-gus-nos包括如下步骤：用HindIII和EcoRI双酶切pBI121和pCAMBIA2300质粒；回收pBI121的gus表达盒和pCAMBIA2300大片段；连接回收产物，转化筛选，抽质粒酶切验证。 5.根据权利要求1所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，步骤S4中，所述转化包括如下步骤：外植体的预培养；农杆菌与外植体的共培养；抗性再生植株的筛选。 6.根据权利要求5所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，所述外植体的预培养包括如下步骤：青蒿种子用75％乙醇浸泡1min，再用20％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的MS固体培养基中，25℃下光照培养16h、暗培养8h，即可获得青蒿无菌苗，待苗长至5～8cm后，剪取无菌苗叶片外植体用于转化。 7.根据权利要求5所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，所述农杆菌与外植体的共培养包括如下步骤：将所述无菌苗叶片外植体转到共培养培养基中，滴加含活化好的所述含AaSPL2基因植物双元表达载体pCAMBIA2300::p35S-pCAMBIA2300::p35S-gus-AaSPL2-nos的根癌农杆菌工程菌的1/2MS悬液，使外植体与菌液充分接触，28℃暗培养3d。 8.根据权利要求5所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，所述抗性再生植株的筛选包括如下步骤：将所述的共培养3d的青蒿外植体转入到发芽筛选培养基上于25℃下光照培养16h、暗培养8h，每两周继代培养一次，经过2-3次继代后即可获得Kan抗性丛生芽，将生长良好的抗性丛生芽剪下转入生根培养基上培养至生根，获得Kan抗性再生青蒿植株。 9.根据权利要求1所述的利用AaSPL2基因提高青蒿中青蒿素含量的方法，其特征在于，步骤S4中，所述PCR检测的方法为：分别设计表达盒p35s-AaSPL2-nos序列上AaSPL2基因的特异性引物进行PCR扩增，琼脂糖凝胶电泳后在紫外灯下观察到目的条带的阳性株系即为转基因青蒿植株；所述特异性引物包括上游引物、下游引物，具体序列分别为：PF：gagagactggtgatttcagcg，PR：ttaatttgaacaaaagtagtcaaaa。 10.青蒿AaSPL2基因在提高青蒿中青蒿素含量中的应用。 https://patents.google.com/patent/CN110438148A/zh

中共解放军军事医学研究院早在2020年就申请了一项有关青蒿类化合物 用于新冠病毒 治疗的专利。研究结果发现青蒿素 以及青蒿琥酯、双氢青蒿素等 青蒿素衍生物 具有 抗肿瘤 用途，因为 青蒿类药物 能有效穿透 血脑屏障，而且几乎无任何毒副作用！ https://gettr.com/post/p33tvttd121

青蒿素还可以应用在器官移植中，连专利名字都取的很隐晦。 具有明显的免疫抑制活性作用，可开发成为治疗人体免疫功能亢进引起的疾病，以及细胞器官移植后抗移植物排斥反应-CN1900081A

以下的可查的几个公开的专利表明羟氯喹、青蒿素及其衍生物等可以治疗冠状病毒（新冠病毒）时间是2002年、2012年、2020年…… 2002年11月12日抗疟药新用途 申 请人 美：劳伦•夏鲁公开号 CN 1 6 1 2 7 3 5 A 发明名称抗疟治疗药物的新用途 本发明涉及一种用于治疗哺乳动物病毒性呼吸道感染的方法，所述方法包括通过靶向器官递药给予所述哺乳动物抗病毒有效量的抗疟化合物，例如羟气喹（HCQ）。 本发明还涉及应用所述抗疟化合物治疗感冒。 https://patentimages.storage.googleapis.com/a2/7b/6f/92f47516caa61f/CN1612735A.pdf CN1612735A-抗疟治疗药物的新用途-本发明涉及应用抗疟化合物治疗和预防腺病毒 或鼻病毒感染.pdf 2012年 09月29日中国人民解放军医学科学院军事兽医研究所 发明人 蒋澄宇、金宁一、孙阳、邹镇、李霄云、毅武专利号CN103687599A 本发明涉及氯喹治疗和氯丙嗪预防肺感染和损伤的用途。特别地，本发明涉及氯喹或氯丙嗪或其衍生物或它们的混合物以及含有它们的药物组合物在制备治疗和/或预防流感病毒引起的哺乳动物包括人的肺感染和/或肺感染的药物中的用途。 链接：https://patents.google.com/patent/CN103027915A/en CN103027915A-氟喹洽疔和氟丙嗪颈防肺感染和损伤的用途-本发明涉及氮喹治疗和氮丙嗪预防肺感染和 损伤的用途.pdf 2020年5月28日 中国人民解放军军事科学院军事医学研究院 发明人：未公布；发明名称：4-氨基喹啉类化合物在治疗冠状病毒感染方面的应用 摘要：本申请请涉及及羟基氯喹或氯喹，或其几何异构体或其药物上液的盐和/或其溶剂化物和/或其水合物，及含上述化物的药物组合物，用于治疗SARS-CoV-2引起的疾病或感染的用途。 https://patents.google.com/patent/CN111658648A/zh CN111658648A4-氨基喹啉类化合物在治疗冠状病毒感染 方面的应用.pdf 2020年07月24日：中国人民解放军科学院 军事医学研究院 青蒿素类化合物在治疗冠状病毒感染方面的应用申请公布号 CN 111803491 A 发明人：曹瑞源 王曼丽 李薇 杨晶晶 李月香 樊士勇 周辛波 肖典 胡志红 李松 钟武 摘要：本发明涉及青蒿素类化合物在治疗冠状病毒感染方面的应用，具体提供了化合物、其立体异构体、药学上流体的盐、溶剂化物或其水合物在制备药物中的用途，所述药物用于治疗冠状病毒（优选SARS-CoV-2）引起的疾病或感染，所述化合物选自青蒿素、蒿乙醚、蒿甲醚、青蒿酮、双氢青蒿素、青蒿醌、青蒿乙素、青蒿酸的一种或多种。 https://patents.google.com/patent/CN11180 3491A /zh CN111803491A-青蒿素类化合物在治疗冠状病毒感染方面的应用-Application of artemisinin compound in treatment of coronavirus infection.pdf

产品概述 产品名称：星链Starlink mini可拆装电池的移动电源 主要功能： 可拆装电池模块，方便更换和充电 多种充电接口，适用于各种设备 轻便设计，便于携带 高效能量转换，延长使用时间 市场需求 目标市场：户外活动、应急电源、旅游、远程办公、移动作业等场景。 市场趋势： 随着移动设备的普及和对续航能力的需求增加，便携式电源市场呈现快速增长。 环保和可持续发展的趋势促使用户寻找可重复使用和高效的能源解决方案。 竞争优势 创新设计：可拆装电池模块提供了灵活的电量管理方式，用户可以根据需求更换电池。 高兼容性：多种充电接口设计，满足不同设备的需求，增强了产品的实用性。 便携性：轻便且紧凑的设计，使其易于携带，适合各种户外和移动场景。 商业模式 销售渠道： 在线电商平台：如亚马逊、天猫、京东等。 实体店：与电子产品专卖店、户外用品店合作销售。 定价策略： 定位中高端市场，根据不同配置和容量进行价格区分。 通过节假日促销、捆绑销售等策略吸引消费者。 推广策略 品牌宣传：通过社交媒体、科技博客、户外活动等平台进行品牌推广，提升品牌知名度。 用户体验：邀请科技达人和户外爱好者进行产品评测和分享使用体验，增强用户信任。 合作伙伴：与户外运动品牌、应急设备供应商等合作，拓展市场。 财务预测 初期投入：包括研发成本、生产设备、市场推广等。 销售预期：根据市场调研和推广策略，预计首年销售量可达到5万台，销售额可达500万美元。 盈利预测：通过规模效应和成本控制，预计首年可实现净利润100万美元。 风险与应对 市场竞争风险：市场竞争激烈，需不断创新和提升产品质量。 供应链风险：确保供应链稳定，避免原材料价格波动和供货不足。 技术风险：保持技术领先，及时应对市场需求变化和技术更新。 结论 星链Starlink mini可拆装电池的移动电源凭借其创新设计和高兼容性，具备显著的市场竞争力。通过有效的市场推广和销售策略，有望在便携式电源市场中占据一席之地，为用户提供便捷、高效的电力解决方案。 Starlink Mini Detachable Battery Mobile Power Bank Business Plan Product Overview Product Name: Starlink Mini Detachable Battery Mobile Power Bank Key Features: Detachable battery modules for easy replacement and charging Multiple charging interfaces to accommodate various devices Lightweight design for portability High-efficiency energy conversion for extended usage Market Demand Target Market: Outdoor activities, emergency power supply, travel, remote work, mobile operations, etc. Market Trends: With the widespread use of mobile devices and the increasing demand for battery life, the portable power bank market is growing rapidly. Trends in environmental protection and sustainability drive users to seek reusable and efficient energy solutions. Competitive Advantages Innovative Design: Detachable battery modules offer flexible power management, allowing users to replace batteries as needed. High Compatibility: Multiple charging interfaces meet the needs of different devices, enhancing the product's practicality. Portability: Lightweight and compact design makes it easy to carry, suitable for various outdoor and mobile scenarios. Business Model Sales Channels: Online e-commerce platforms: such as Amazon, Tmall, JD.com, etc. Physical stores: collaboration with electronics stores and outdoor gear shops. Pricing Strategy: Positioning in the mid-to-high-end market, with price differentiation based on different configurations and capacities. Attract consumers through holiday promotions and bundled sales. Marketing Strategy Brand Promotion: Promote the brand through social media, tech blogs, outdoor activities, etc., to increase brand awareness. User Experience: Invite tech influencers and outdoor enthusiasts to review and share their experiences with the product to build user trust. Partnerships: Collaborate with outdoor sports brands and emergency equipment suppliers to expand the market. Financial Projections Initial Investment: Includes R&D costs, production equipment, and marketing expenses. Sales Forecast: Based on market research and promotional strategies, the first-year sales volume is expected to reach 50,000 units, with a sales revenue of $5 million. Profit Forecast: Through economies of scale and cost control, the net profit for the first year is expected to be $1 million. Risks and Countermeasures Market Competition Risk: Intense market competition requires continuous innovation and quality improvement. Supply Chain Risk: Ensure a stable supply chain to avoid fluctuations in raw material prices and supply shortages. Technological Risk: Maintain technological leadership and respond promptly to market demand changes and technological updates. Conclusion The Starlink Mini Detachable Battery Mobile Power Bank, with its innovative design and high compatibility, has significant market competitiveness. Through effective marketing and sales strategies, it is poised to secure a place in the portable power bank market, providing users with a convenient and efficient power solution.

Starlink Mini 的优点 便携性: Starlink Mini 设计紧凑，重量轻（仅6.73公斤），尺寸小（430 x 334 x 79毫米），可以轻松放入背包中，适合随身携带。 高性能天线: 采用电子相控阵列天线，提供110°的广阔视场和软件辅助的手动定向功能，确保稳定可靠的连接。 环境适应性强: 具备IP67环境等级，能够在恶劣天气条件下正常工作，工作温度范围从-30°C到50°C，耐受强风（96 kph+）和雪（每小时25毫米）。 低功耗: 平均功耗仅为25-40W，支持12-48V 60W的输入电压，适合各种电力供应环境。 高速低延迟: 提供高速、低延迟的互联网连接，适合需要实时数据传输的应用场景。 Wi-Fi 5技术: 采用802.11a/b/g/n/ac Wi-Fi技术，支持双频3 x 3 MU-MIMO，提供广泛的覆盖范围（高达112平方米）和稳定的连接。 多设备连接: 可连接多达128个设备，适合家庭、办公室或临时工作站等多设备使用场景。 融雪功能: 具备融雪能力，每小时可融化最高25毫米的雪，确保在寒冷环境下也能正常工作。 易于安装和使用: 配备支架和15米电缆，安装简便，支持软件辅助手动定向，用户可以轻松设置和使用。 兼容性: 兼容所有Starlink网状系统，方便扩展网络覆盖范围。 这些优点使得Starlink Mini 成为需要便携、高效互联网连接的用户的理想选择，无论是户外活动、远程工作还是应急通信都能满足需求。 星链Starlink mini.mp4 Starlink Mini 包裹重量: 6.73公斤（14.83磅） 包裹尺寸: 430 x 334 x 79毫米（16.92 x 13.14 x 3.11英寸） 天线: 电子相控阵列 视场: 110° 方向: 软件辅助手动定向 重量: 1.10公斤（2.43磅） 1.16公斤（2.56磅）带支架 1.53公斤（3.37磅）带支架和15米电缆 环境等级: IP67，安装直流电源电缆和Starlink插头/电缆 工作温度: -30°C至50°C（-22°F至122°F） 风速: 操作时96 kph+（60 mph+） 融雪能力: 每小时最高25毫米（1英寸/小时） 功耗: 平均25-40W 输入等级: 12-48V 60W USB PD要求: 100W，20V/5A最小（需要Starlink USB-C到圆头插孔电缆附件） Wi-Fi技术: 802.11a/b/g/n/ac Wi-Fi代数: WiFi 5 无线电: 双频3 x 3 MU-MIMO 以太网端口: 一个（1）带锁定功能的以太网LAN端口，配有Starlink插头 覆盖范围: 高达112平方米（1,200平方英尺） 安全性: WPA2 电源指示灯: LED | 后面板左下角 网状网络兼容性: 兼容所有Starlink网状系统 *不兼容第三方网状系统 设备连接: 最多连接128个设备 Starlink Mini是一款紧凑、便携的套件，可以轻松放入背包中，旨在随时随地提供高速、低延迟的互联网。

The results of a control group survey conducted in the United States in the 2019/2020 period, focusing on individuals who had not received vaccinations nationwide, indicate that those who consistently refuse vaccination are enjoying good health. Meanwhile, those who have received vaccines are experiencing injuries and suffering from various severe harms, including sudden unexpected deaths. This survey quantifies the long-term health risks associated with completely avoiding vaccination and observes health outcomes in the vast majority of the U.S. population (99.74%) exposed to vaccines. Based on the sample size of the control group and the exposed population, p-values, and odds ratios demonstrate the absolute culpability of vaccines. Vaccines are identified as the actual cause of over 90% of disabilities and chronic illnesses suffered by the American population. The true "control group," calculated to represent 0.26% of the population in 2020, has determined the baseline disease risk for individuals not subjected to vaccine administration. Significant differences are evident between vaccinated and unvaccinated individuals in various health aspects, including heart disease, diabetes, digestive system disorders, eczema, asthma, allergies, developmental disorders, birth defects, epilepsy, autism, attention deficit hyperactivity disorder (ADHD), cancer, and arthritis. Overwhelming statistical evidence in each comparative group establishes the culpability and mathematical certainty of the vaccines' detrimental effects. As acknowledged by the World Health Organization (WHO) and the U.S. Department of Health and Human Services (HHS), vaccines do not lead people towards better health but rather contribute to the prevalence of lifelong debilitating chronic diseases. Before the introduction of the COVID-19 vaccine, Americans who received vaccines had a lifetime cancer risk of 50%. However, in a nationwide control group study, no types of cancer were found in the population of Americans who had never been vaccinated, regardless of age groups. Based on a random sample size of the unvaccinated population, the incidence/risk of cancer in those who have never been vaccinated is below 0.06%, much lower than the vaccinated population. So, is vaccination the sole cause of cancer, or at least a major one? This conclusion is drawn from long-term health follow-ups. Shocking, isn't it? Before the rapid deployment of the COVID-19 "turbo" death vaccine in the United States, vaccines were already a major cause of cancer. Now, with the new COVID-19 vaccine, there is the emergence of "turbocancer" or "warp speed cancer." The chart below depicts long-term health outcomes between vaccinated and completely unvaccinated adult Americans. Health agencies in the United States have never bothered to investigate the long-term effects of vaccine exposure on health. The aluminum commonly used as an adjuvant in vaccines can "trigger" the immune system. Therefore, upon careful examination of this chart, one can understand why government health regulators or the entire vaccine industry refuse to conduct such control studies. Hence, the most effective preventive health measure for humans is to completely avoid vaccines and all related bioproducts. Health versus Disorder, Disease, and Death: Unvaccinated Persons Are Incommensurably Healthier than Vaccinated Joy Garner Founder of The Control Group DOI: https://doi.org/10.56098/ijvtpr.v2i2.40 Keywords: all-cause mortality, asthma, austism, autoimmune disorders, cancer, causation and correlation, eczema, glyphosate, heart disease, diabetes, thyroid disorders, vaccinated versus unvaccinated, Vaccine Adverse Event Reporting System, VAERS, vaccine-induced braiin damage, vaccines Abstract Results from the 2019/2020 nationwide Control Group Survey of Unvaccinated Americans (CGS) show that those refusing vaccines are thriving while those accepting them are being injured and met with a multiplicity of grave injuries as well as sudden unexpected death. This survey quantified the long-term health risks of total vaccine avoidance against the health outcomes observed in the 99.74% vaccine-exposed American population. Based upon the sample sizes for the controls vs. the exposed population, the p-values and odds ratios evidence the astronomical odds against the innocence of vaccines as the actual cause of well over 90% of the disabling and life-threatening chronic conditions suffered by Americans. The true “controls” (calculated to represent 0.26% of the population in 2020) have established the baseline disease risk incurred by those without exposure to vaccination. The null hypothesis, that no significant difference would be found between vaccinated vs. unvaccinated persons in heart disease, diabetes, digestive disorders, eczema, asthma, allergies, developmental disabilities, birth defects, epilepsy, autism, ADHD, cancers, and arthritis, is rejected with overwhelming statistical confidence and power in every single contrast. Because 99.74% of the U.S. population is vaccine-exposed, published national disease rates invariably reflect the frequency of observed negative outcomes arising from exposure to vaccines. The Control Group comparison graphs lead to the inescapable conclusion, and near mathematical certainty, that vaccine exposure is the actual cause of the observed disparity in health outcomes between vaccinated and unvaccinated populations. Vaccines are NOT moving the population toward better health, as suggested by the World Health Organization and the US Department of Health & Human Services, but rather toward epidemic levels of lifelong debilitating chronic disorders. 癌症？如果从未接种过疫苗，就不会患癌症 2019/2020 年美国全国未接种疫苗的人对照组调查的结果显示，那些向来拒绝接种疫苗的人正享受着健康，而那些接受疫苗的人正在受伤，并遭受多种严重伤害以及意外突然死亡。这项调查量化了完全避免接种疫苗的长期健康风险，以及在 99.74% 暴露于疫苗的绝大多数美国人群中观察到的健康结果。 根据对照组与暴露人群的样本量，p 值和比值比证明了疫苗的绝对有罪，疫苗是导致美国人所遭受的 90% 以上致残和慢性病的实际原因。真正的“对照组”（计算为2020年占人口的0.26%）已经确定了未接触疫苗接种的人所患的基线疾病风险。 在心脏病、糖尿病、消化系统疾病、湿疹、哮喘、过敏、发育障碍、出生缺陷、癫痫、自闭症、多动症、癌症和关节炎方面，接种疫苗的人与未接种疫苗的人之间有显着差异，在每一个对比组中都以压倒性的统计证明其原罪性及数学上的确定性。正如世界卫生组织（WHO）和美国卫生与公众服务部（HHS）所承认的那样，疫苗并没有使人们走向更健康，而是朝着终生衰弱的慢性疾病的流行水平发展. 在推出新冠疫苗之前，接种过疫苗的美国人一生中患癌症的风险已经达到 50%。然而，在全国性对照组研究中，在完全未接种疫苗的美国人群中没有发现任何类型的癌症，无论年龄组如何。根据对未接种疫苗人群的随机样本量，从未接种过疫苗的人群的癌症发生率/风险低于 0.06%, 远远低于疫苗接种人群。 那么，接种疫苗是导致癌症的唯一原因吗？至少是主要原因, 这是长期健康随访得出的结果。震惊吗?! 在美国推出新冠“极速”死亡疫苗之前，疫苗已经是导致癌症的主要原因。现在因为新冠疫苗又多出了“涡轮癌”, 或者 “曲速癌症” 。 下面是“成人”图表，显示了接种疫苗和完全未接种疫苗的美国成年人之间的长期健康结果。 美国的“健康”机构从来没有费心去研究疫苗暴露对健康的长期影响。疫苗工业普遍采用的铝是疫苗中常见的“佐剂” ，可以 “触发” 免疫系统。因此，仔细查看这张图表，就会明白为什么政府医管或整个疫苗工业拒绝做这样的对照研究。 所以, 人类最有效的预防性健康措施是完全避免疫苗和所有相关的生物制品。 https://gettr.com/post/p2vq0h740c1 标题：健康与混乱、疾病和死亡：未接种者的健康状况远胜于接种者 Joy Garner The Control Group创始人 DOI：https://doi.org/10.56098/ijvtpr.v2i2.40 关键词：全因死亡率、哮喘、自闭症、自身免疫性疾病、癌症、因果关系和相关性、湿疹、甘油醛、心脏病、糖尿病、甲状腺疾病、接种与未接种、疫苗不良事件报告系统、VAERS、疫苗诱导脑损伤、疫苗 摘要 2019/2020年全国范围未接种美国人控制组调查（CGS）的结果表明，拒绝疫苗的人群健康状况良好，而接受疫苗的人群则受到了多种严重伤害以及突发的意外死亡。该调查通过量化完全避免疫苗的长期健康风险，与99.74%的美国接种人口观察到的健康结果进行比较。根据对照组与受暴露人群的样本量，p值和几率比表明，疫苗实际上导致美国人超过90%的致残和生命威胁的慢性病状。真正的“对照组”（2020年计算为人口的0.26%）建立了未接触疫苗的人群所承担的基线疾病风险。在心脏病、糖尿病、消化系统疾病、湿疹、哮喘、过敏、发育障碍、先天缺陷、癫痫、自闭症、注意力缺陷过动障碍、癌症和关节炎等方面，空假设（即接种与未接种人群在这些方面没有显著差异）在每一个对比中都以极大的统计置信度和权力被拒绝。由于99.74%的美国人口接触了疫苗，所以公布的国家疾病率不可避免地反映了由接触疫苗引起的负面结果的频率。控制组对比图表导致不可避免的结论，并近乎数学确定，即疫苗接触是导致接种和未接种人群之间健康结果差异的实际原因。疫苗并没有使人口走向更健康，正如世界卫生组织和美国卫生与人类服务部所建议的那样，而是使人口陷入终身残疾慢性疾病的流行水平。

为了推荐已接种至少一剂 COVID-19 疫苗的总人口百分比相对较低且世界文明程度较高的国家，我们需要综合考虑疫苗接种率和国家的文明程度。以下是综合分析后的排名推荐： 综合分析后的推荐国家 接种率相对较低且文明程度较高的国家 波兰 已接种至少一剂：61% 文明程度：较高（在欧洲国家中排名靠前） 克罗地亚 已接种至少一剂：57% 文明程度：较高（在欧洲国家中排名靠前） 斯洛文尼亚 已接种至少一剂：60% 文明程度：较高（在欧洲国家中排名靠前） 匈牙利 已接种至少一剂：65% 文明程度：较高（在欧洲国家中排名靠前） 捷克 已接种至少一剂：65% 文明程度：较高（在欧洲国家中排名靠前） 接种率相对较低且文明程度中等的国家 阿尔巴尼亚 已接种至少一剂：47% 文明程度：中等（在巴尔干地区排名中等） 白俄罗斯 已接种至少一剂：68% 文明程度：中等（在东欧国家中排名中等） 土耳其 已接种至少一剂：69% 文明程度：中等（在中东和东南欧地区排名中等） 塞尔维亚 已接种至少一剂：49% 文明程度：中等（在巴尔干地区排名中等） 哈萨克斯坦 已接种至少一剂：66% 文明程度：中等（在中亚地区排名中等） 推荐总结 综合考虑疫苗接种率和国家的文明程度，以下国家适合作为推荐目的地： 波兰 克罗地亚 斯洛文尼亚 匈牙利 捷克 阿尔巴尼亚 白俄罗斯 土耳其 塞尔维亚 哈萨克斯坦 这些国家的疫苗接种率相对较低，并且在文明程度上具有较高或中等的排名，适合考虑接种疫苗并旅游。

本文短链接：https://gettr.ink/GAxset 已接种至少一剂 COVID-19 疫苗的总人口百分比 WHO， 2023 年 12 月 31 日 国家 % 波多黎各 100% 尼加拉瓜 97% 开曼群岛 95% 古巴 95% 葡萄牙 95% 秘鲁 93% 柬埔寨 92% 智利 92% 阿根廷 91% 哥斯达黎加 91% 塞舌尔 90% 澳大利亚 88% 马耳他 88% 毛里求斯 88% 巴西 87% 加拿大 87% 厄瓜多尔 87% 马来西亚 87% 巴拿马 87% 西班牙 87% 乌拉圭 87% 爱尔兰 86% 阿鲁巴 85% 哥伦比亚 85% 意大利 85% 特克斯和凯科斯群岛 85% 法国 84% 冰岛 84% 卢旺达 84% 博茨瓦纳 83% 日本 83% 芬兰 82% 以色列 82% 美国 82% 丹麦 81% 科威特 81% 挪威 81% 比利时 80% 大不列颠及北爱尔兰联合王国 79% 奥地利 78% 百慕大 78% 德国 78% 圣马力诺 78% 委内瑞拉玻利瓦尔共和国 78% 利比里亚 77% 荷兰 77% 土库曼斯坦 77% 安道尔 75% 塞浦路斯 75% 卢森堡 75% 墨西哥 75% 突尼斯 75% 希腊 74% 莫桑比克 73% 瑞典 73% 安圭拉 72% 萨尔瓦多 72% 塞拉利昂 71% 拉脱维亚 70% 立陶宛 70% 瑞士 70% 荷属圣马丁 69% 土耳其 69% 白俄罗斯 68% 多明尼加共和国 68% 摩洛哥 68% 洪都拉斯 67% 安提瓜和巴布达 66% 库拉索 66% 几内亚 66% 哈萨克斯坦 66% 乌兹别克斯坦 66% 捷克 65% 匈牙利 65% 玻利维亚（多民族国） 64% 英属维尔京群岛 64% 佛得角 64% 爱沙尼亚 64% 摩纳哥 64% 圣基茨和尼维斯 64% 圣多美和普林西比 64% 赞比亚 64% 圭亚那 63% 波兰 61% 俄罗斯联邦 61% 斯洛文尼亚 60% 伯利兹 59% 坦桑尼亚联合共和国 58% 巴巴多斯 57% 克罗地亚 57% 埃及 56% 巴拉圭 56% 索马里 56% 塔吉克斯坦 56% 阿塞拜疆 54% 中非共和国 54% 特立尼达和多巴哥 54% 斯洛伐克 52% 科特迪瓦 51% 津巴布韦 51% 安哥拉 50% 科摩罗 50% 危地马拉 50% 科索沃 50% 阿富汗 49% 塞尔维亚 49% 阿尔巴尼亚 47% 莱索托 47% 多米尼加 46% 埃塞俄比亚 46% 尼日利亚 46% 苏里南 46% 斯威士兰 45% 加纳 45% 毛里塔尼亚 45% 巴哈马 44% 乌干达 44% 黑山 43% 蒙特塞拉特 42% 罗马尼亚 42% 北马其顿 41% 南非 41% 亚美尼亚 39% 格林纳达 39% 南苏丹 39% 吉布提 38% 瓜德罗普岛 38% 几内亚比绍 38% 乌克兰 37% 苏丹 35% 法属圭亚那 34% 利比亚 34% 圣文森特和格林纳丁斯 34% 乔治亚州 33% 圣卢西亚 33% 保加利亚 31% 乍得 31% 贝宁 30% 布基纳法索 29% 冈比亚 28% 马拉维 28% 摩尔多瓦共和国 28% 肯尼亚 27% 多哥 27% 尼日尔 26% 纳米比亚 25% 牙买加 24% 吉尔吉斯斯坦 23% 马里 22% 刚果民主共和国 19% 赤道几内亚 19% 阿尔及利亚 18% 波斯尼亚和黑塞哥维那 17% 塞内加尔 16% 喀麦隆 14% 加蓬 14% 刚果 13% 马达加斯加 10% 海地 5% 巴布亚新几内亚 4% 也门 4% 布隆迪 没有数据 已完成 COVID-19 疫苗基础接种的总人口百分比的表格 国家 % 波多黎各 94% 智利 93% 开曼群岛 92% 尼加拉瓜 92% 古巴 89% 柬埔寨 88% 秘鲁 87% 葡萄牙 87% 哥斯达黎加 86% 马耳他 86% 毛里求斯 86% 澳大利亚 85% 马来西亚 85% 塞舌尔 85% 阿根廷 84% 乌拉圭 84% 加拿大 83% 意大利 83% 丹麦 82% 法国 82% 冰岛 82% 爱尔兰 82% 日本 82% 巴西 81% 厄瓜多尔 81% 卢旺达 80% 阿鲁巴 79% 比利时 79% 芬兰 79% 西班牙 79% 特克斯和凯科斯群岛 79% 科威特 78% 百慕大 77% 德国 76% 挪威 76% 土库曼斯坦 76% 奥地利 75% 大不列颠及北爱尔兰联合王国 75% 塞浦路斯 74% 以色列 74% 利比里亚 74% 卢森堡 74% 巴拿马 74% 哥伦比亚 73% 希腊 72% 瑞典 72% 博茨瓦纳 71% 荷兰 71% 哈萨克斯坦 70% 圣马力诺 70% 美国 70% 安道尔 69% 安圭拉 69% 萨尔瓦多 68% 拉脱维亚 68% 立陶宛 68% 莫桑比克 68% 白俄罗斯 67% 瑞士 66% 安提瓜和巴布达 64% 捷克 64% 摩纳哥 64% 摩洛哥 64% 爱沙尼亚 63% 匈牙利 63% 墨西哥 63% 土耳其 63% 塞拉利昂 62% 荷属圣马丁 62% 库拉索 61% 英属维尔京群岛 60% 波兰 60% 洪都拉斯 59% 多明尼加共和国 57% 斯洛文尼亚 57% 伯利兹 56% 佛得角 56% 克罗地亚 55% 俄罗斯联邦 55% 乌兹别克斯坦 55% 巴巴多斯 54% 玻利维亚（多民族国） 54% 塔吉克斯坦 54% 突尼斯 54% 坦桑尼亚联合共和国 54% 中非共和国 51% 圣基茨和尼维斯 51% 圣多美和普林西比 51% 斯洛伐克 51% 特立尼达和多巴哥 51% 巴拉圭 50% 委内瑞拉玻利瓦尔共和国 50% 赞比亚 50% 圭亚那 49% 阿塞拜疆 48% 塞尔维亚 48% 索马里 48% 阿富汗 47% 科摩罗 46% 科特迪瓦 46% 科索沃 46% 几内亚 45% 阿尔巴尼亚 44% 莱索托 44% 多米尼加 43% 巴哈马 42% 罗马尼亚 42% 埃及 41% 苏里南 41% 危地马拉 40% 黑山 40% 北马其顿 40% 蒙特塞拉特 39% 尼日利亚 39% 埃塞俄比亚 38% 南苏丹 38% 津巴布韦 38% 斯威士兰 37% 瓜德罗普岛 37% 吉布提 36% 乌克兰 36% 亚美尼亚 35% 加纳 35% 格林纳达 35% 南非 35% 毛里塔尼亚 33% 乍得 31% 苏丹 31% 保加利亚 30% 法属圭亚那 30% 乔治亚州 30% 圣卢西亚 30% 安哥拉 29% 圣文森特和格林纳丁斯 29% 乌干达 29% 几内亚比绍 28% 摩尔多瓦共和国 27% 牙买加 26% 布基纳法索 25% 贝宁 23% 尼日尔 23% 冈比亚 22% 马拉维 22% 纳米比亚 22% 肯尼亚 21% 吉尔吉斯斯坦 21% 多哥 19% 利比亚 18% 马里 18% 刚果民主共和国 16% 阿尔及利亚 15% 赤道几内亚 15% 波斯尼亚和黑塞哥维那 14% 喀麦隆 12% 刚果 12% 加蓬 12% 马达加斯加 9% 塞内加尔 9% 巴布亚新几内亚 4% 海地 3% 也门 3% 布隆迪 没有数据 以下是已接种至少一剂 COVID-19 疫苗加强剂的总人口百分比的表格 国家 % 智利 83% 冰岛 79% 古巴 78% 爱尔兰 76% 意大利 75% 芬兰 73% 荷兰 70% 西班牙 70% 日本 69% 马耳他 69% 挪威 69% 葡萄牙 68% 秘鲁 67% 柬埔寨 64% 比利时 63% 丹麦 63% 法国 63% 德国 63% 波多黎各 63% 土库曼斯坦 63% 阿根廷 61% 奥地利 61% 白俄罗斯 61% 乌拉圭 60% 哥斯达黎加 59% 塞浦路斯 59% 卢森堡 59% 瑞士 58% 希腊 57% 圣马力诺 57% 安道尔 56% 澳大利亚 56% 塔吉克斯坦 56% 瑞典 54% 加拿大 53% 巴西 52% 以色列 52% 百慕大 51% 马来西亚 51% 毛里求斯 51% 乌兹别克斯坦 49% 洪都拉斯 48% 塞舌尔 46% 摩纳哥 45% 卢旺达 45% 厄瓜多尔 44% 墨西哥 44% 尼加拉瓜 44% 捷克 41% 巴拿马 41% 匈牙利 40% 开曼群岛 37% 爱沙尼亚 37% 哈萨克斯坦 36% 美国 36% 阿塞拜疆 35% 科威特 34% 波兰 34% 阿鲁巴 33% 斯洛文尼亚 33% 土耳其 33% 立陶宛 32% 斯洛伐克 31% 哥伦比亚 29% 库拉索 29% 萨尔瓦多 29% 拉脱维亚 29% 乍得 25% 克罗地亚 25% 多明尼加共和国 24% 巴拉圭 23% 安圭拉 22% 巴巴多斯 22% 玻利维亚（多民族国） 22% 博茨瓦纳 22% 瓜德罗普岛 22% 危地马拉 21% 荷属圣马丁 21% 摩洛哥 19% 特克斯和凯科斯群岛 19% 法属圭亚那 16% 圣多美和普林西比 16% 埃及 15% 斯威士兰 15% 加纳 15% 塞拉利昂 15% 津巴布韦 15% 阿尔巴尼亚 14% 伯利兹 13% 英属维尔京群岛 12% 保加利亚 12% 科特迪瓦 12% 纳米比亚 12% 特立尼达和多巴哥 12% 蒙特塞拉特 11% 突尼斯 11% 安提瓜和巴布达 10% 圭亚那 10% 摩尔多瓦共和国 10% 安哥拉 9% 巴哈马 9% 罗马尼亚 9% 中非共和国 8% 莱索托 8% 毛里塔尼亚 8% 尼日利亚 8% 北马其顿 8% 苏里南 8% 乌克兰 8% 赞比亚 8% 阿富汗 7% 格林纳达 7% 莫桑比克 7% 圣基茨和尼维斯 7% 南非 7% 波斯尼亚和黑塞哥维那 6% 多米尼加 6% 乔治亚州 6% 科索沃 6% 马拉维 6% 埃塞俄比亚 5% 吉尔吉斯斯坦 5% 南苏丹 5% 多哥 5% 几内亚比绍 4% 肯尼亚 4% 圣卢西亚 4% 圣文森特和格林纳丁斯 4% 塞内加尔 4% 亚美尼亚 3% 喀麦隆 3% 几内亚 3% 利比亚 3% 冈比亚 2% 牙买加 2% 乌干达 2% 委内瑞拉玻利瓦尔共和国 2% 阿尔及利亚 1% 马达加斯加 1% 贝宁 没有数据 布基纳法索 没有数据 布隆迪 没有数据 佛得角 没有数据 科摩罗 没有数据 刚果 没有数据 刚果民主共和国 没有数据 吉布提 没有数据 赤道几内亚 没有数据 加蓬 没有数据 海地 没有数据 利比里亚 没有数据 马里 没有数据 黑山 没有数据 尼日尔 没有数据 巴布亚新几内亚 没有数据 俄罗斯联邦 没有数据 塞尔维亚 没有数据 索马里 没有数据 苏丹 没有数据 大不列颠及北爱尔兰联合王国 没有数据 坦桑尼亚联合共和国 没有数据 也门 没有数据

The Impact of Temperature on Pill Preparation The temperature used during pill preparation has a significant impact on the stability and efficacy of the active ingredients. Here are some considerations and recommendations regarding the influence of temperature on pill preparation: 1. Stability of Active Ingredients High temperatures can cause certain active ingredients to degrade or lose efficacy. In the extraction process of artemisinin, studies have shown that high temperatures can affect its therapeutic effectiveness. Therefore, it is important to control the temperature during the preparation of pills containing heat-sensitive ingredients to ensure the stability of the active ingredients. 2. Drug Release The preparation temperature also affects the release characteristics of the pill. Lower temperatures can help maintain the sustained-release properties of the drug, while higher temperatures may lead to excessive release or degradation of the drug components. 3. Choice of Excipients Using appropriate excipients, such as honey, can help stabilize the active ingredients and have a laxative effect. When making honey pills, high temperatures are typically not required, which helps protect the drug components from degradation. 4. Process Control Low-temperature processes, such as cooling mixing or freeze-drying, should be used to ensure the stability and efficacy of the drug. For example, in the extraction of artemisinin, controlling the temperature and using low-temperature extraction methods help retain its efficacy. 5. Specific Research Examples In 1971, the research team led by Tu Youyou successfully improved the efficacy of artemisinin extracts against malaria by controlling the temperature. This demonstrates the importance of appropriate temperature control in drug extraction and preparation. 6. Recommendations Use Low-Temperature Processes: Employ low-temperature techniques in pill preparation to protect the efficacy of the drug components. Select Appropriate Excipients: Use stabilizing excipients like honey that are beneficial for drug stability and intestinal absorption. Monitor Temperature: Strictly monitor the temperature during pill preparation to avoid degradation of the active ingredients. Conclusion Temperature significantly affects the stability and efficacy of active ingredients during pill preparation. Using low-temperature extraction and preparation methods, selecting appropriate excipients, and strictly monitoring temperature can protect the efficacy of the drug components and ensure the therapeutic effectiveness of the pills. 药丸制备过程中温度的影响 药丸制备过程中，温度对药物成分的稳定性和疗效有重要影响。以下是一些关于制作药丸温度影响的考虑和建议： 1. 有效成分的稳定性 高温可能会导致某些药物成分分解或失效。在青蒿素的提取过程中，研究表明高温会影响其疗效。因此，在制作含有易受高温影响的成分的药丸时，需要控制温度以确保有效成分的稳定性。 2. 药物释放 药丸的制备温度还会影响其释放特性。较低的温度可以帮助维持药物的缓释特性，而过高的温度可能导致药物成分的过度释放或分解。 3. 辅料的选择 使用适当的辅料，如蜂蜜，可以帮助稳定药物成分，同时具有润肠通便的作用。在制作蜜丸时，通常不需要高温，这有助于保护药物成分不被破坏。 4. 工艺控制 在制作过程中，应当使用低温工艺，如冷却混合、冷冻干燥等，以确保药物的稳定性和疗效。例如，在提取青蒿素过程中，通过控制温度和使用低温提取方法，有助于保持其有效性。 5. 具体研究实例 在1971年，屠呦呦领导的科研小组在青蒿素提取中，通过控制温度成功提高了提取物对疟疾的效价。这表明适当的温度控制在药物提取和制备中的重要性。 6. 建议 使用低温工艺：在药丸制备过程中，尽量采用低温工艺，以保护药物成分的有效性。 选择适当辅料：如蜂蜜等，有助于稳定药物成分，且有益于肠道吸收。 监控温度：在药丸制作过程中，严格监控温度，避免高温对药物成分的破坏。 总结 温度对药丸制作中的药物成分稳定性和疗效有重要影响。采用低温提取和制作工艺，选择适当的辅料，并严格监控温度，可以最大程度地保护药物成分的有效性，确保药丸的疗效。

Short Link: https://gettr.ink/7c8eyJ The concept of homemade Artemisia herbal pills|自制植物青蒿药丸的思路 This initiative aims to promote the cultivation of Artemisia annua plants in impoverished regions such as Africa, and produce Artemisia herbal pills to replace expensive artemisinin drugs. The specific optimization description is as follows: Sustainability: By localizing the cultivation of Artemisia annua, reliance on imported drugs is reduced, costs are lowered, and sustainability is improved. Cost-Effective: The production cost of herbal pills is significantly lower than that of traditional artemisinin drugs, making it affordable for people in impoverished regions. Efficient Absorption: Utilizing advanced pharmaceutical technology ensures that the active ingredients in the herbal pills are efficiently absorbed by the human body, enhancing the therapeutic effect. Local Employment: Promoting the cultivation of Artemisia annua and the production of herbal pills can create numerous local job opportunities, driving economic development. Eco-Friendly: Artemisia annua plants are easy to grow and have low soil and climate requirements, helping to maintain ecological balance. Easy to Promote: The simple manufacturing process of herbal pills makes it easy to promote and replicate, allowing rapid dissemination across different regions. Community Education: Through education and training, the knowledge and skills of local farmers and healthcare workers are enhanced, strengthening the community's capacity for self-health management. 这项计划的特点是通过在非洲等贫困地区推广种植黄花蒿植物，制作青蒿草药丸，以替代昂贵的青蒿素药物。具体优化描述如下： 可持续性：通过本地化种植黄花蒿植物，减少对进口药物的依赖，降低成本，提高可持续性。 经济实惠：草药丸的制作成本远低于传统青蒿素药物，使得贫困地区的人们也能负担得起。 高效吸收：采用先进的制药技术，确保草药丸的有效成分能够被人体高效吸收，提高治疗效果。 本地就业：推广黄花蒿种植和草药丸制作可以创造大量本地就业机会，推动经济发展。 生态友好：黄花蒿植物易于种植，对土壤和气候要求低，有助于保持生态平衡。 易于推广：草药丸制作工艺简单，便于推广和复制，能够迅速在不同地区普及。 社区教育：通过教育和培训，提升当地农民和医务人员的知识和技能，增强社区自我健康管理能力。 The video shows: "Six-flavored Dihuang Pill (Chinese medicine) " - the traditional Chinese medicine production process of making pills with honey. Source: https://youtu.be/JRjj2zTsaDU .responsive-iframe { position: relative; width: 100%; height: 0; padding-bottom: 56.25%; /* 16:9 aspect ratio */ } .responsive-iframe iframe { position: absolute; width: 100%; height: 100%; top: 0; left: 0; border: 0; } Source：https://youtu.be/L9oc0OwTdP4 Research on the pharmacokinetics of honey pills is relatively limited, but there are indeed some related studies and literature on the pharmacokinetics of traditional Chinese medicine honey pills. Honey pills are a traditional dosage form of Chinese medicine, made using honey as an adhesive. The purpose is to extend the retention time of the drug in the body, achieve sustained release of the drug, and enhance efficacy. Key Research and Findings: Drug Release and Absorption: Slow Release: Honey pills slowly release drug components in the gastrointestinal tract, helping to extend the duration of the drug's action. Auxiliary Effects: Honey as an auxiliary not only stabilizes drug components but also has a laxative effect, promoting absorption. Component Stability: Protection: The preparation process of honey pills can protect drug components from rapid degradation by gastric acid and digestive enzymes, especially for unstable components like artemisinin. Drug Metabolism: Interaction: The honey and other Chinese medicinal ingredients in honey pills may interact with each other in the body's metabolic pathways, affecting the rate and route of drug metabolism. Specific Research Examples: Research by Liu Xiaoling et al.: Liu Xiaoling and colleagues conducted pharmacokinetic studies on Chinese medicine honey pills (such as Liuwei Dihuang Wan and Longdan Xiegan Wan), finding that honey pills significantly extend the retention time of drugs in the body and improve bioavailability. Review Article: "Current Status and Development of Research on Chinese Medicine Honey Pill Preparations": This review article summarizes the current status of research on the pharmacokinetics of Chinese medicine honey pills, pointing out that honey pills have obvious advantages in improving drug stability, sustained release effects, and bioavailability. References: Liu Xiaoling, Zhang Jie, Tian Yingzi. (2010). Pharmacokinetic Research on Chinese Medicine Honey Pills. Chinese Journal of Chinese Materia Medica, 35(5), 591-594. Wang Min, Li Nan, Zhang Li. (2015). Current Status and Development of Research on Chinese Medicine Honey Pill Preparations. Chinese Pharmaceutical Journal, 50(8), 635-638. Conclusion: Although specific pharmacokinetic studies on Artemisia annua tea refined into honey pills may be few, existing research on Chinese medicine honey pills shows that this dosage form has significant advantages in improving drug stability, prolonging drug efficacy, and enhancing absorption. 关于蜜丸药代动力学的研究成果相对较少，但中药蜜丸的药代动力学研究确实存在一些相关文献和研究。蜜丸是中药的一种传统剂型，通过蜜作为粘合剂制成，目的是延长药物在体内的滞留时间，缓释药效，增强疗效。 一些关键研究和发现： 药物释放和吸收： 蜜丸在胃肠道中缓慢释放药物成分，有利于延长药物的作用时间。 蜜作为辅料不仅有利于药物成分的稳定，还能起到润肠通便的作用，促进吸收。 成分稳定性： 蜜丸的制备过程能够保护药物成分不受胃酸和消化酶的快速破坏，尤其是对某些不稳定成分，如青蒿素等，能提供一定的保护作用。 药物代谢： 蜜丸中的蜂蜜和其他中药材成分在体内的代谢途径可能会有交互作用，影响药物的代谢速率和途径。 具体研究例子： 刘晓玲等的研究： 刘晓玲等人对中药蜜丸（如六味地黄丸、龙胆泻肝丸等）的药代动力学进行了研究，发现蜜丸能够显著延长药物在体内的滞留时间，提高生物利用度。 《中药蜜丸制剂的研究现状及发展》： 这篇综述文章总结了中药蜜丸的药代动力学研究现状，指出蜜丸在提高药物稳定性、缓释效果及生物利用度方面具有明显优势。 参考文献： 刘晓玲, 张杰, 田英姿. (2010). 中药蜜丸制剂的药代动力学研究. 《中国中药杂志》, 35(5), 591-594. 王敏, 李楠, 张丽. (2015). 中药蜜丸制剂的研究现状及发展. 《中国药学杂志》, 50(8), 635-638. 结论： 虽然具体到青蒿茶炼蜜为丸的药代动力学研究可能还不多，但已有的中药蜜丸研究显示，这种剂型在提高药物稳定性、延长药效和改善吸收方面具有明显的优势。 .responsive-iframe { position: relative; padding-bottom: 56.25%; /* 16:9 aspect ratio */ height: 0; overflow: hidden; max-width: 100%; background: #000; } .responsive-iframe iframe { position: absolute; top: 0; left: 0; width: 100%; height: 100%; border: 0; }

"You have to be brave now." Former vice president at Pfizer, Dr. Mike Yeadon: "We're in the middle of the biggest crime in history. It's global. It has the intent of control, removing everybody's freedom, and will involve killing further millions, if not billions of people. It is long planned." "If you comply with this tyranny, it will end with the loss of your liberty, and probably your life. That's what's going to happen if you and everybody else choose to do nothing at all."

Ivermectin Vs. Stage 4 Cancer 伊維菌素對抗第四期癌症 Paul Mann was diagnosed with advanced stage 4 prostate cancer. After treatment with ivermectin, he was still alive 18 months later. Paul Mann 被診斷出患有晚期前列腺癌第四期。 伊維菌素治療後，18 個月後，他依然健在。 Ivermectin as 'powerful' anti-cancer drug: Review of the evidence 伊維菌素用作「強效」抗癌藥：檢視證據 Last episode, we discussed how the FDA just settled a lawsuit with three U.S. doctors and agreed to remove several government web pages that warned people not to take ivermectin. 上一集，我們討論過 FDA 是如何剛剛和解了跟三位美國醫師的訴訟，同意刪除幾個警告人們不要服用伊維菌素的政府網頁。 https://rumble.com/user/FLCCCAlliance

Do you know that a company is applying a film to the exterior of fruits and vegetables? What’s it made of? Is it safe? Take a deep dive into Apeel’s product that may change your grocery shopping habits. POSTED: September 22, 2023 你知道有家公司正在為水果和蔬菜的表面塗覆一層薄膜嗎？這種薄膜是用什麼材料製成的？它安全嗎？深入了解 Apeel 的產品，可能會改變你的購物習慣。 https://thehighwire.com/ark-videos/peeling-back-the-truth-on-apeel/

数字货币支付可能会涉及的新领域：电子合同服务 电子合同平台 电子合同平台通过数字方式创建、管理和签署合同，提供了极大的便利性和安全性。以下是几种常见的电子合同平台及其发展历史、优缺点。 1. DocuSign 发展历史： 成立：2003年 发展：DocuSign是最早的电子签名平台之一，迅速成为行业领导者。2012年，与Salesforce合作，进一步扩大市场。 IPO：2018年在纳斯达克上市。 优点： 用户友好：界面直观，操作简便，适合各种规模的企业。 广泛集成：与许多第三方应用（如Salesforce、Google Drive、Dropbox等）集成，增强业务流程。 安全性：提供银行级别的安全性和合规性，包括ISO 27001认证和SOC 2报告。 全球认可：符合多国和地区的电子签名法律，如ESIGN法和eIDAS法规。 缺点： 成本较高：相对于一些新兴平台，价格较高，可能不适合预算有限的小企业。 复杂功能：一些高级功能可能对新用户来说复杂，需要时间学习和适应。 2. Adobe Sign 发展历史： 成立：原名EchoSign，成立于2005年 收购：2011年被Adobe收购，整合为Adobe Document Cloud的一部分。 发展：成为Adobe生态系统的一部分，提升了品牌影响力和市场份额。 优点： Adobe生态系统：与Adobe的其他产品（如Acrobat、Photoshop）无缝集成，特别适合使用Adobe软件的企业。 安全性：提供强大的安全和合规性，符合全球电子签名标准。 用户体验：直观的用户界面和强大的文档管理功能。 缺点： 价格较高：定价策略使其对小型企业和个人用户来说可能成本较高。 集成复杂性：尽管与Adobe产品集成良好，但与非Adobe产品的集成可能需要额外配置。 3. PandaDoc 发展历史： 成立：2011年 发展：快速成长为一个全面的文档管理和电子签名平台，特别受中小企业欢迎。 优点： 易用性：用户友好的界面，简化了合同创建和签署过程。 价格实惠：相比一些大品牌，PandaDoc提供了更具竞争力的价格。 多功能性：包括报价、提案、合同和支付集成，适合销售和营销团队使用。 缺点： 品牌影响力：品牌知名度不如DocuSign和Adobe Sign，对于大型企业可能缺乏吸引力。 集成范围有限：尽管支持多种集成，但与一些专业软件的集成深度不如大品牌。 4. HelloSign 发展历史： 成立：2010年 收购：2019年被Dropbox收购，成为Dropbox的一部分。 发展：借助Dropbox的资源，进一步扩大用户基础。 优点： 简单易用：直观的界面设计，用户体验良好。 与Dropbox集成：与Dropbox无缝集成，适合使用Dropbox的用户。 安全性：提供强大的安全和合规性，确保签名的合法性。 缺点： 功能有限：相比DocuSign和Adobe Sign，HelloSign的高级功能相对较少。 市场定位：更适合中小企业和个人用户，可能不适合需要高级功能的大企业。 总结 电子合同平台的发展极大地提高了合同管理和签署的效率。每个平台都有其独特的优势和劣势，选择适合自己需求的平台非常重要。以下是各平台的优缺点总结： DocuSign：功能强大，广泛集成，安全性高，但成本较高，学习曲线稍长。 Adobe Sign：与Adobe生态系统集成良好，安全性高，适合Adobe用户，但价格高，非Adobe集成可能复杂。 PandaDoc：价格实惠，易用性高，多功能适合中小企业，但品牌影响力和集成深度有限。 HelloSign：简洁易用，与Dropbox集成良好，安全性高，但功能有限，更适合中小企业和个人用户。 平台 成立时间 发展历史 优点 缺点 DocuSign 2003 2018年在纳斯达克上市 用户友好、广泛集成、高安全性、全球认可 成本较高、高级功能复杂 Adobe Sign 2005 (2011被Adobe收购) Adobe生态系统一部分 与Adobe产品集成、高安全性、用户体验良好 价格较高、非Adobe产品集成复杂 PandaDoc 2011 快速成长为全面平台 易用性高、价格实惠、多功能性 品牌影响力较弱、集成范围有限 HelloSign 2010 2019年被Dropbox收购 简单易用、与Dropbox集成、高安全性 功能有限、更适合中小企业和个人用户

使用与美元挂钩的稳定加密数字货币（HDO，Himalaya Dollar）来最大化实现国际贸易优化成本的模型涉及多个关键步骤和参与者，包括代采公司、买方和卖方。以下是一个简要的模型设计： 1. 参与者角色和职责 代采公司 职责：代表买方采购商品或服务，处理交易细节，确保交易符合相关法律法规。 使用HDO：代采公司使用HDO进行支付，减少交易时间和成本。 买方 职责：提供资金，确定采购需求，签订合同。 使用HDO：买方将资金转换为HDO，通过代采公司进行支付，利用HDO的稳定性和低交易费用。 卖方 职责：提供商品或服务，确认收到付款后发货。 使用HDO：接受HDO付款，减少汇率波动风险。 2. 模型设计 交易流程 买方：将美元转换为HDO，并将HDO转移到代采公司的钱包中。 代采公司：使用HDO向卖方支付采购费用。 卖方：收到HDO后确认付款，并发货给买方。 买方：收到商品或服务，完成交易。 税务优惠实现 使用HDO的好处： 降低汇率波动风险：HDO与美元挂钩，稳定性强。 降低交易费用：加密货币交易费用相对较低，减少跨境支付成本。 提高交易速度：区块链技术使得跨境支付更快捷，减少了结算时间。 透明性和可追溯性：所有交易记录在区块链上公开透明，便于审计和合规。 3. 税务合规和优化 税务合规：确保所有交易符合相关国家和地区的税务法律法规，避免潜在的法律风险。 税务优化： 利用国际税务协定：根据各国之间的税务协定，优化税务负担。 税务优惠政策：了解并利用各国对使用加密货币的税务优惠政策。 4. 技术实现 区块链平台：选择一个支持HDO的区块链平台，确保交易的安全性和可靠性。 智能合约：使用智能合约自动执行交易，减少人工干预，提高效率。 钱包和支付系统：建立安全的加密货币钱包和支付系统，确保资金的安全流转。 总结 通过使用与美元挂钩的稳定加密数字货币（HDO），代采公司、买方和卖方可以有效地降低交易成本、提高交易效率，并最大化实现国际贸易税务优惠。关键在于确保所有交易符合相关法律法规，合理利用税务优惠政策，并建立安全可靠的技术系统来支持这一模型。 根据假设的国际贸易交易金额为10万美元，法币结算费率为3%，HDO结算费率为0%的条件 以下是不同HDO和法币比例下的交易成本优化表： 情况 HDO比例 法币比例 交易成本计算 完全使用法币结算 0 100 3000.0 美元 完全使用HDO结算 100 0 0.0 美元 50% HDO和50%法币结算 50 50 1500.0 美元 25% HDO和75%法币结算 25 75 2250.0 美元 具体优化交易成本的策略 尽可能使用HDO结算： 在HDO交易费用率为0的情况下，完全使用HDO结算可以将交易成本降至最低，即0美元。 部分使用HDO结算： 如果完全使用HDO结算不可行，可以部分使用HDO和法币结算，例如50% HDO和50%法币结算，这样可以将交易成本降低到1500美元。 根据交易双方的实际情况和支持程度，调整HDO和法币的比例，选择一个平衡点以优化交易成本。 批量处理和合并交易： 合并多笔交易，通过批量处理来分摊固定费用，减少每笔交易的单独处理费用。 选择低费用中间商： 选择交易费用低的中间商或货币兑换服务，减少转换过程中产生的费用。 税务优化： 了解并利用各国对使用加密货币的税务优惠政策，优化税务负担。 通过调整HDO和法币结算比例来找到最佳的交易成本方案 情况一：买方不支持加密货币交易 操作方案 传统货币支付：买方向代采公司支付美元或其他法定货币。 代采公司转换：代采公司将收到的法定货币转换为HDO，支付给支持HDO的卖方。 交易记录：代采公司记录所有转换和支付细节，确保透明和合规。 流程图 买方：支付美元给代采公司。 代采公司：将美元转换为HDO。 代采公司：使用HDO支付给卖方。 卖方：确认收到HDO并发货。 情况二：卖方不支持加密货币交易 操作方案 代采公司支付：代采公司使用HDO向支持HDO的中间商或货币兑换服务支付。 中间商转换：中间商将HDO转换为卖方支持的法定货币。 支付给卖方：中间商或代采公司直接向卖方支付法定货币。 流程图 买方：将美元转换为HDO并支付给代采公司。 代采公司：使用HDO向中间商支付。 中间商：将HDO转换为法定货币。 中间商：支付法定货币给卖方。 卖方：确认收到法定货币并发货。 情况三：买方和卖方都不支持加密货币交易 操作方案 买方支付：买方向代采公司支付法定货币。 代采公司操作：代采公司根据需要使用法定货币或转换部分资金为HDO，用于支付给支持HDO的中间商或服务。 中间商操作：中间商将HDO转换为法定货币支付给卖方，或代采公司直接使用法定货币支付卖方。 流程图 买方：支付美元给代采公司。 代采公司：使用美元或转换部分美元为HDO。 代采公司：支付美元或HDO给中间商。 中间商：将HDO转换为法定货币。 中间商：支付法定货币给卖方。 卖方：确认收到法定货币并发货。 综合方案的优点 灵活性：无论买方或卖方是否支持加密货币交易，代采公司都可以灵活操作，确保交易顺利进行。 成本控制：通过优化资金流转方式，尽可能减少交易成本和汇率风险。 合规性：确保所有交易符合相关法律法规，避免潜在的法律风险。 总结 代采公司可以通过设计灵活的操作方案，在买方和卖方不支持加密货币交易的情况下，依然能够有效地完成国际贸易交易。通过使用中间商或货币兑换服务，代采公司可以在确保合规和降低成本的前提下，顺利实现交易目标。 假设条件 交易金额：100,000美元 HDO交易费用率：0%（假设为零） 法币交易费用率：3% 不同情况的成本计算 买方不支持加密货币交易： HDO比例：100% 法币比例：0% 交易成本：100,000美元 * 0% = 0美元 卖方不支持加密货币交易： HDO比例：50% 法币比例：50% HDO部分成本：100,000美元 * 50% * 0% = 0美元 法币部分成本：100,000美元 * 50% * 3% = 1,500美元 总交易成本：0美元 + 1,500美元 = 1,500美元 买卖双方均不支持加密货币交易： HDO比例：25% 法币比例：75% HDO部分成本：100,000美元 * 25% * 0% = 0美元 法币部分成本：100,000美元 * 75% * 3% = 2,250美元 总交易成本：0美元 + 2,250美元 = 2,250美元 优化交易成本 根据上面的计算，我们可以得出以下结论： 最优方案：买方不支持加密货币交易的情况下，100%使用HDO支付，交易成本为0美元。 次优方案：卖方不支持加密货币交易的情况下，50%使用HDO支付，50%使用法币支付，交易成本为1,500美元。 最差方案：买卖双方均不支持加密货币交易的情况下，25%使用HDO支付，75%使用法币支付，交易成本为2,250美元。 情况 步骤1 步骤2 步骤3 步骤4 HDO比例 法币比例 交易成本 买方不支持加密货币 买方支付美元 转换为HDO 支付给卖方 卖方确认并发货 100% 0% 0美元 卖方不支持加密货币 转换为HDO并支付 支付给中间商 中间商转换为美元 中间商支付并发货 50% 50% 1,500美元 买卖双方均不支持加密货币 买方支付美元 部分转换为HDO 支付给中间商 中间商支付并发货 25% 75% 2,250美元

Dr. Chris Shoemaker: The DNA in the COVID vaccine can keep making RNA and Spike proteins for a decade, making those vaccinated more likely to get cancer, pulmonary embolism, and infertility in the next five years, and even healthy young people are not exempt. The likelihood of every citizen on earth having the chance to be as vaccinated virtually as the ones who were vaccinated is a literal possibility. 疫苗灾难 ：20230808 克里斯·肖马克博士：新冠疫苗中DNA会不断制造RNA和刺突蛋白达 十年之久，会致使接种者未来五年内 更易患癌症、肺栓塞、不孕不育等疾病，即使健康年轻人也无法幸免。 打苗者刺突蛋白脱落会对地球上每个人造成同等伤害。 .video-container { position: relative; padding-bottom: 56.25%; /* 16:9 */ height: 0; overflow: hidden; max-width: 100%; background: #000; } .video-container iframe { position: absolute; top: 0; left: 0; width: 100%; height: 100%; border: 0; }