Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用

The effect of Caspase-8 and Bcl-2 in the apoptosis induced by PIG11 protein in human hepatocellular carcinoma HepG2 cells

目的:利用本实验室已建立PIG11蛋白稳定低表达的HepG2细胞株和PIG11蛋白稳定高表达HepG2细胞株,研究Caspase-8和Bcl-2在PIG11诱导HepG2细胞凋亡中的作用,进一步探讨PIG11基因表达对HepG2细胞凋亡的机制。方法:细胞培养,分组:HepG2细胞、pLXSN-PIG11-HepG2(pLXSN-PIG11转染建立PIG11蛋白高表达HepG2细胞)、pLXSN-HepG2(pLXSN空载体转染HepG2细胞)、miR-PIG11-HepG2(miRNA干扰PIG11蛋白低表达HepG2细胞)、miR-HepG2(干扰空载体HepG2细胞)。Hoechst33342染色荧光和PI染色流式细胞仪检测各组细胞凋亡情况。Western blot检测Caspase-8蛋白、Bcl-2蛋白的表达情况。结果:Hoechst 33342染色荧光显微镜下pLXSN-PIG11-HepG2细胞组可见核染色质浓缩,核碎片,荧光增强,以及凋亡小体。PI染色流式细胞仪检测结果显示:HepG2细胞、miR-PIG11-HepG2细胞、miR-HepG2细胞、pLXSN-PIG11-HepG2细胞、pLXSN-HepG2细胞的凋亡率分别为5.72%±0.81%、1.34%±0.71%、5.10%±0.40%、34.83%±2.29%、5.34%±0.60%。PIG11高表达的pLXSN-PIG11-HepG2细胞组凋亡率比其它各组增高(P<0.01),PIG11低表达的miR-PIG11-HepG2细胞组凋亡率降低(P<0.01)。Western blot检测结果显示PIG11高表达的pLXSN-PIG11-HepG2细胞Caspase-8蛋白表达上调,Bcl-2蛋白表达下调(P<0.01),PIG11低表达的miR-PIG11-HepG2细胞Caspase-8蛋白的表达下调,Bcl-2蛋白表达上调(P<0.01)。结论:PIG11蛋白高表达能诱导HepG2细胞凋亡,其机制可能与Caspase-8蛋白及Bcl-2蛋白表达相关。

Objective：Using the HepG2 cell line low expressing PIG11gene and the HepG2 cell line high expressing PIG11gene which was constructed before by our research group,to research on the function of PIG11 gene in liver cancer cell and the role of which to promote apoptosis.Explore the mechanism of expression PIG11 gene for HepG2 cells lead to apoptosis.Methods：To culture five groups of cell ： HepG2 cell,pLXSN-PIG11-HepG2 cell,pLXSN-HepG2 cell,miR-PIG11-HepG2 cell,miR-HepG2 cell,and compare apoptosis of five groups of cell using Hoechst 33342 dyeing fluorescence,flow cytometer scans（FACS）,and Western blot analysis.Results：Chromatin condensation,nuclear fragmentation and apoptotic body were observed in pLXSN-PIG11-HepG2 cell group.Flow cytometer scans showed that the apoptosis ratios of HepG2,miR-PIG11-HepG2,miR-HepG2,pLXSN-PIG11-HepG2,pLXSN-HepG2 were 5.72%±0.81%,1.34%±0.71%,5.10%±0.40%,34.83%±2.29%,5.34%±0.60%.The apoptosis ratio of HepG2 cell with PIG11 gene high expression was higher while that of HepG2 cell with PIG11 gene low expression was the opposite than others.The expression of Caspase-8 was higher while that of HepG2 cell with PIG11 gene low expression were lower than others.And the expression of Bcl-2 was lower while that of HepG2 cell with PIG11 gene low expression was higher than others.Conclusion：The apoptosis ratio of HepG2 cell with PIG11 gene high expression was higher.HepG2 cells apoptosis induced by PIG11 was related to Caspase-8 and Bcl-2.