| Objective:This experimental is about study the effecting on activity by low-dose triptolide(TPL)combined with cisplatin(DDP)on cisplatin-resistant human ovarian cancer cells(SKOV3/DDP)in vitro and observe triptolide and cisplatin whether have a synergistic antitumor effect,preliminary explored its underlying mechanism of antitumor and the mechanism of synergistic antitumor effect of SKOV3/DDP cells.Methods:1.SKOV3/DDP cells subculture:SKOV3/DDP cells subculture:In constant temperature incubator with 37?,5%CO2 and saturated hnmidity,with the medium containing 10%FBS and 100u/ml penicillin and streptomycin RPMI-1640 cultured SKOV3/DDP cells,When the cells were filled with 80%-90% at the bottom of the culture bottle,the conventional digestion,centrifugation and replacement of the culture media were then inoculated according to the proportion of 1:3.2.The influence after the DDP done with SKOV3/DDP cells by MTT assay:Different concentrations of DDP(0.3125?g/ml,0.625?g/ml,1.25?g/ml,2.5?g/ml,5?g/ml,10?g/ml,20?g/ml,40?g/ml)respectively in SKOV3/DDP cells for 24 h,48h,72 h after using the OD value was detected by MTT.The effects of DDP on the proliferation of SKOV3/DDP cells at different concentrations and different action time were observed,calculated the inhibition rate,IC50 value and IC10 value.3.The influence after the TPL done with SKOV3/DDP cells by MTT assay:Different concentrations of TPL intervention(0.5ng/ml,1ng/ml,2ng/ml,4ng/ml,8ng/ml,16ng/ml,32ng/ml,64ng/ml)in SKOV3/DDP cells for 24 h,48h,72 h after using the OD value was detected by MTT.The effects of TPL on the proliferation of SKOV3/DDP cells at different concentrations and different action time were observed,calculated the inhibition rate,IC50 value and IC10 value.4.The influence after the DDP combined with TPL done with SKOV3/DDPcells by MTT assay:Group TPL(concentration: 0.5ng/ml,2ng/ml,8ng/ml)and group DDP(concentration: 0.3125?g/ml,1.25?g/ml,5?g/ml)combined group and single drug group the concentration of two two Coalition.SKOV3/DDP cells were treated with 24 h MTT method was used to detect the OD,calculated the inhibition rate of SKOV3/DDP cells in each group,and according to the Chou-Talalay method,the combined index of two drugs was calculated.5.The effect of TPL combined with DDP on apoptosis of SKOV3/DDP cells by flow cytometry:SKOV3/DDP cells were cultured for 24 h by control groups(no drug),TPL groups(0.5ng/ml),DDP groups(10?g/ml)and combinative groups of DDP(10?g/ml)and TPL(0.5ng/ml).The rate of apoptosis was measured by flow cytometry using Annexin V-FITC/PI staining.6.The effect of TPL combined with DDP on the expression of Smac protein,Survivin protein and Caspase-3 protein in SKOV3/DDP cells was detected by blot Western method:SKOV3/DDP cells were cultured for 24 h by control groups(no drug),TPL groups(0.5ng/ml),DDP groups(10?g/ml)and combinative groups of DDP(10?g/ml)and TPL(0.5ng/ml).The expression of Smac,Survivin,Caspase-3 Was determined by Western blot.Results:1.From the growth curve of SKOV3/DDP cells,after 24-72 hours is the logarithmic growth phase cells,determined to take the role of 24-72 hours cells as target cells.2.DDP concentration is high,the role of time longer,on cell proliferation inhibition more.The IC50 after 24 h was 14.197?g/ml,after 48 h was 6.5?g/ml,after72 h was 2.994?g/ml.The IC10 after 24 h was 0.597?g/ml.The IC10 after 24 h was0.102?g/ml.3.TPL concentration is high,the role of time longer,on cell proliferation inhibition more.The IC50 after 24 h was 14.197ng/ml,after 48 h was 6.5ng/ml,after72 h was 2.994ng/ml.The IC10 after 24 h was 0.597ng/ml.4.Low,medium and high DDP concentration group(0.3125?g/ml,1.25?g/ml,5?g/ml)and low,middle,high TPL concentration group(0.5ng/ml,2ng/ml,8ng/ml)and each concentration group pairwise combined to form the combined group were treated SKOV3/DDP cells for 24 h.The results showed that the proliferation inhibition rate of every combined group was higher than the corresponding single drug group,the difference was statistically significant(P < 0.01),DDP and TPL combined with index CI < 1.5.The control group,TPL(0.5ng/ml)group,DDP(10?g/ml)group and TPL(0.5ng/ml)combined with cisplatin(10?g/ml)group were treated with DDP 24 h after,mean cell apoptosis rates were control group 4.66% + 0.93%,TPL group was 8.33%+ 0.96%,DDP group was 19.82% + 2.56%,DDP combined with TPL group is24.73% + 2.01%.The cell apoptosis rates of TPL group,DDP group and combined group increase significantly compared to control group,the difference was statistically significant(P < 0.01).Combined group have a higher cell apoptosis rates compared to TPL group and DDP group,the difference was statistically significant(P< 0.01).6.SKOV3/DDP cells were cultured for 24 h by control groups(no drug),TPL groups(0.5ng/ml),DDP groups(10?g/ml)and combinative groups of DDP(10?g/ml)and TPL(0.5ng/ml),Western Blot showed that TPL group,DDP group and combined group all have a higher expression of Smac protein and Caspase-3 protein compared to control group,the difference was statistically significant(P < 0.01).Combined group compared with TPL group and DDP group of Smac protein and Caspase-3protein up-regulated,Survivin protein expression was decreased,there are statistically significant(P<0.05)compared with control group,TPL group compared with DDP group of Smac protein,the difference was not statistically significant(P>0.05).Conclusion:1.Both TPL and DDP have inhibitory effect on the proliferation of cisplatin resistant human ovarian cancer cells(SKOV3/DDP)in vitro,the drug concentration and the treatment time is longer,the more obvious inhibitory effect on cells,and0.5ng/ml low-dose TPL can enhance the inhibitory effect of DDP on SKOV3/DDP cells.2.Both TPL and DDP can induce apoptosis of SKOV3/DDP,and 0.5ng/ml low-dose TPL can enhance the apoptosis of SKOV3/DDP cells induced.3.TPL induced apoptosis of SKOV3/DDP cells,the mechanism of cisplatin resistance and sensitization may be related to the upregulation of pro-apoptotic protein Smac and Caspase-3,and decreased the expression of anti-apptotic protein Survivin. |
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