Application of the qinghaosu in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation|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
Entry posted by Roger in Artemisinin Patent｜青蒿素专利
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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
flow cytomery is carried out immediately.
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.
the violent Vortex of most high speed 5 seconds, 4 DEG C of 12,000-16,000g are centrifuged 5 minutes.
Block buffer is sucked, the primary antibody after dilution is added.
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
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CN201910349857.7A2019-04-28Application of the qinghaosu in the hepatoma Hep G 2 cells drug candidate of preparation treatment EMT activation
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)
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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
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