| BackgroundCurrently,ovarian cancer has been the gynecological tumor with the highest mortality rate,which threats women's health seriously.The resistance and adverse reactions problems about the drugs for the treatment of ovarian cancer still exist.To find effective drugs with less adverse reaction is becoming a hot spot of the study of ovarian cancer.ObjectivesTo observe the effect of thymoquinone and the combination of cisplatin and thymoquinone to the cell cycle of apoptosis of ovarian cancer SK-OV-3 cell,and further explore its mechanism from the angle of the expressing change of Bcl-2 and Bax.Methods1.Looking for the effective concentration of cisplatin and thymoquinone which can inhibit the growth of ovarian cancer SK-OV-3 cells.Cell proliferation-toxic kits(cell counting kit-8,CCK 8)was used to test the effective inhibition concentration of cisplatin and thymoquinone after the intervention of 24 h,48h and 72 h.2.Lodide third organism(propidium iodide,PI)and flow cytometry instrument was used to detect the effect of cisplatin,thymoquinone and the combination of cisplatin and thymoquinone on cell cycle of SK-OV-3.3.Through the Annexin V-FTTC fluorescence and PI marking the nucleic acid by application of phosphate ester acyl serine(Annexin V)valgus analysis,and by using flow cytometry instrument detection of effects of cisplatin,thymoquinone and thymoquinone combined cisplatin on SK-OV-3 cells apoptosis.4.To detect the effects of cisplatin,thymoquinone and thymoquinone combined cisplatin on levels of mRNA of Bcl-2 and Bax in SK-OV-3 cells,by the application of real-time fluorescent quantitative PCR detection of cisplatin(Real-time PCR)technology5.To detect the effects of cisplatin,thymoquinone and thymoquinone combined cisplatin on protein expression levels of Bcl-2 and Bax in SK-OV-3 cells,by the application of Western Blot method(Western Blot).Results1.5? mol/L,10? mol/L,15? mol/and 20? mol/L of cisplatin was respectively used to intervent SK-OV-3 cell.After the intervention of 24 h,48h and 72 h,cell proliferation-toxicity was detected by CCK-8,and the OD value of the SK-OV-3 cell of the was lower in the 15? mol/and 20? mol/L group,compared to 5? mol/L and 10?mol/L group,with statistical significance.So we choose 15 ? mol/L cisplatin as the effective concentration to intervent SK-OV-3 cells,and 48 h after intervention as the observation time point.2.10 ? mol/L,15 ? mol/L,20 ? mol/L and 25 ? mol/L of thymoquinone was respectively used to intervent SK-OV-3 cell.After the intervention of 24 h,48h and 72 h,cell proliferation-toxicity was detected by cck-8,and the OD value of the SK-OV-3 cell of the was significantly lower in the 20? mol/L and 25? mol/L group,compared to 10?mol/L and 15 ? mol/L group,with statistical significance.So we choose 20umol/L thymoquinone as the effective concentration to intervent SK-OV-3 cells,and 48 h after intervention as the observation time point.3.The s SK-OV-3 cell was intervented in four ways respectively: none drug was given(the control group),dealing with 15? mol/L cisplatin(the Cis group),processing with 20? mol/L thymoquinone(the TQ group),and managing with 15? mol/L cisplatin and 20? mol/L of thymoquinone(the Cis+TQ group).The proliferation-toxicity of the ovarian carcinoma cells was detected by CCK-8,after the intervention of 24 h,48h and 72 h.It shows that the OD value of the ovarian cancer cells was significantly lower in the TQ group and Cis+TQ,comparing with the control group(P<0.05)and Cis group at 24 h after the intervention(P < 0.05).And the OD value of the Cisgroup is significantly lower than the control group(P<0.05);At 48 h after intervention,the order of OD value of groups of cells are the Control group>Cis group >Cis+TQ group,and each the difference between the two groups showed statistical significance(P<0.05);72 hour after the intervention,OD value of the ovarian cancer cells in Cis,TQ group and Cis+TQ group were significantly lower than the control group(P<0.05).Besides,OD value in the Cis +TQ group was less than the Cis and TQ groups,differences are statistically significant(P <0.05).4.It was found in Cell cycle detection the control group had a significantly lower percentage of S phase than cis percentage,the TQ and cis + TQ group(P < 0.05)in 48 h after drug intervention.5.It was observed in cell apoptosis detection that cell apoptosis rate was significantly lower than the control group Cis,TQ and Cis+TQ group(P <0.05);And apoptosis rate of Cis+TQ group is significantly higher than Cis and TQ group(P<0.05)at 48 h after drug intervention.6.Real-Time PCR detection found in 48 h after drug treatment,the mRNA of the Bcl-2 in SK-OV-3 cell of the Control group is significantly higher than Cis,TQ and Cis+TQ group(P<0.05).At this time for SK-OV-3 in cells.The level of mRNA expression of SK-OV-3 cells indicated that there was a significant decrease in SK-OV-3 cells in Control group as compared to Cis group,TQ group and Cis+TQ group(p<0.05).Moreover,there was a significant increase in Cis+TQ group as compared to Cis group and TQ group(p<0.05).7.Western blot analysis indicated that after 48 hours of treatment,for level of Bcl-2 and Bax,there is a significant increase in SK-OV-3 cells in Control group as compared to Cis group,TQ group and Cis+TQ group(p<0.05).Moreover,there is a significant decrease in Cis+TQ group as compared to Cis group and TQ group(p<0.05).Conclusion1.TQ group,Cis+TQ group and Cis group can inhibit cell proliferation of ovarian cancer effectively,and the combined effect of Cis+TQ group is better than simple effect of either of TQ group and Cis group.2.TQ group,Cis+TQ group and Cis group inhibit cell proliferation by extending S period.3.TQ group,Cis+TQ group and Cis group promote cell apoptosis of ovarian cancer,and the combined effect ofCis+TQ group is better than simple effect of either of TQ group and Cis group.4.TQ group,Cis+TQ group and Cis group promote cell apoptosis by inhibiting expression of Bcl-2 and promoting expression of Bax,and the combined effect of Cis+TQ group is better than simple effect of either of TQ group and Cis group. |
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