| Objective 1.Different concentrations of vitamin K2(VK2)and berberine towards hepatocellular carcinoma cell line Hep-G2,and to explore their effects and mechanism of inhibition of cell proliferation,induction of cell apoptosis.2.Intervening hepatocellular carcinoma cell line Hep-G2 with vitamin K2 and berberine mixture,further to study whether it is more effective on the combination of two drugs to inhibit the proliferation of hepatoma cells.3.According to the analysis of the experimental with berberine which is an extract of traditional Chinese medicine,whether it can enhance the curative effect of vitamin K2,in order to explore the effect of traditional Chinese medicine in the treatment of hepatocellular carcinoma.MethodsTo investigate the inhibitory effect of berberine on the proliferation of Hep-G2 cells with the methods of CCK8.Taking the half inhibitory concentration,that is IC50 value,as the final concentration of drug combination group to treat cells for 48 hours.Flow cytometry detected the cell proliferation and apoptosis of the cells affected by VK2 ?berberine and the combination of two drugs;Western blotting and RT-PCR detected the protein expression level of BTG2,anti proliferative gene,and CyclinD1,the expression of BTG2 mRNA and CyclinD1 mRNA after intervening by VK2?berberine and the combination of two drugs.Results 1.the proliferiton of Hep-G2 cell lines were inhibited by VK2 in a dose and time dependent manner,and compared with the control group,the difference was statistically significant(p<0.01).VK2 blocks the cell cycle in the G1 phase,and promotes cell apoptosis,and the rate of apoptosis is proportional to the time of VK2 action(P<0.05).The expression of BTG2 protein gradually increased,while the expression of Cyclin D1 protein decreased as the time of VK2 treatment,compared with the control group,the differential expression of BTG2 and CyclinD1 protein was significant(P<0.01).The enhancement of BTG2 mRNA expression did not change significantly with the prolongation of VK2(P>0.05),and compared with the control group,the difference was not significant(P>0.05),while the decrease of CyclinD1 mRNA expression changed obviously(P<0.05),and there was a significant difference between the control group(P<0.01).2.The proliferiton of Hep-G2 cell lines were inhibited by berberine in a dose and time dependent manner,and compared with the control group,the difference was statistically significant(P<0.05).Berberine blocks the cell cycle in the G1 phase,and promotes cell apoptosis,and the rate of apoptosis is proportional to the time of berberine action(P<0.05).The expression of BTG2 protein gradually increased,while the expression of cyclin Cyclin D1 protein decreased as the time of berberine treatment,compared with the control group,the differential expression of BTG2 and CyclinD1 protein was significant(P<0.01).The expression of BTG2 mRNA was gradually enhanced with the extension of time of Berberine effection,but the difference was not obvious(P>0.05),while the expression of CyclinD1 mRNA was gradually decreased with the time of berberine intervention(P<0.05),and the difference was significant compared with the control group(P<0.01).3.After two kinds of drugs were mixed,Hep-G2 cells were detected in the liver cancer cells,and the inhibition rate of 48 h was slightly decreased from 24h-48 h,while after 48 hours,the inhibition rate increased gradually.Further analysis showed that compare with the VK2 group and berberine group,the difference is significant(P<0.01).In the combined group,the cell cycle was blocked in the G1 phase which had the effect of promoting apoptosis,and the apoptosis rate was proportional to the time of the combined group(P<0.05);Further analysis showed that after three groups of drug interventing cells,the proportion of G0/G1 phase cells increased gradually,the proportion of S phase cells decreased gradually,and the proportion of G2 phase cells was not obvious.The expression of BTG2 protein was significantly increased with the prolongation of the combined group(P<0.01),at the same time,the expression of Cyclin D1 protein was not significantly reduced at 24h-48h(P>0.05),However,the expression was significantly decreased in other time(P<0.01),and the general trend was gradually decreased with the prolongation of the combined group.Compared with the control group,the protein expression of BTG2 and CyclinD1 were significantly different(P<0.01);Further analysis shows that,although no significant difference of protein expression between VK2 group and berberine group,but compared the protein expression of BTG2 and Cyclin D1 of combined group with VK2 group and berberine,the difference is significant(P<0.01),and the protein expression of the three groups respectively are compared with the control group,it also shows a significant differences(P<0.01).The expression of BTG2 mRNA from 24h-72 h did not show time dependence,but compared with the VK2 group and berberine group,the expression was significantly increased at each time period(P<0.01),and compared with the control group,there is a significant difference(P<0.01).The expression of CyclinD1 mRNA was not significantly decreased in 24h-48 h time(P>0.05),but decreased significantly in other time(P<0.01).The general trend was that the expression of the combined group was decreased gradually,and the difference was significant compared with the control group(P<0.01).Conclusion 1.VK2 and berberine can inhibit the proliferation of Hep-G2 cells,promote cell apoptosis,which is time-dependent.2.Inhibitory effect of VK2 on proliferation of hepatocellular carcinoma Hep-G2 cells will be enhanced after the combination of VK2 and Berberine.3.VK2 and berberine can provide a new way for the treatment of liver cancer,but the two drugs are mixed,the effect will be more obvious,it would be a better method for the treatment of liver cancer. |
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