bax过表达对乙醇诱导的肝癌细胞凋亡的促进作用

Overexpression of bax enhances ethanol-induced apoptosis in hepatoma cells

目的探讨促凋亡基因bax过表达对乙醇诱导的原发性肝细胞癌(简称肝癌)细胞凋亡的调节作用。方法用脂质体介导的基因转染法将含有人bax cDNA的噬菌粒表达载体pBK-bax质粒导入人肝癌细胞系HCC-9204细胞中,同时设转染pBK-CMV空载体的和未转染的HCC-9204细胞对照。通过用G-418筛选后获得转入目的基因的细胞克隆。用免疫细胞化学ABC法、图像定量分析和Westert blot法检测bax蛋白在转染bax或空载体前后细胞中的表达情况。用6%乙醇分别作用于上述细胞6h后,MTT法检测细胞杀伤率,TUNEL染色法和流式细胞仪DNA含量分析法检测细胞凋亡的发生程度。结果转染后用G-418持续筛选2周,获得了导入bax或空载体的G-418抗性的细胞克隆。转染bax基因的细胞对bax蛋白的表达强度明显强于转染空载体或未转染的细胞,而转染空载体与未转染细胞的bax蛋白表达强度无明显差异。经低浓度乙醇作用后,转染bax的肝癌细胞的细胞杀伤率、TUNEL指数和亚二倍体凋亡峰比例均明显高于转染空载体或未转染的细胞。结论bax蛋白过表达能够促进低浓度乙醇诱导的肝癌HCC-9204细胞凋亡。

Purpose To explore the regulative function of pro-apoptotic gene bax overexpression in ethanol-induced apoptosis of primary hepatocellular carcinoma （HCC） cells. Methods The plasmid of bacteriophague expression vector pBK-bax, which contains human bax cDNA, was transduced into HCC cell line HCC-9204 cells with Lipid-mediated gene transfection method, and at the same time the HCC-9204 cells transfected with empty pBK-CMV plasmid and non-transfected HCC-9204 cells were taken as control. The cell clones, into which objective genes had been transfected, were obtained after being screened with G-418. The expression of bax protein in the cells prior to and after transfected with bax or empty vector was detected with immunocytochemical ABC method, image quantitative analysis and Westorn blot. After the above cells were affected by 6% ethanol for 6 h, the killing rates of the cells were detected with MTT method. The degree of apoptosis was detected with TUNEL staining and DNA contents analysis by flow cytometry. Results The G-418-resistant cell clones, which had been transfected with bax or empty vector were obtained by screening continually with G- 418 for 2 w after transfection. There was stronger expression of bax protein in the cells transfected with bax gene than in those transfected with empty vector or non-transfected cells. There was no significant difference between the cells transfected with empty vector and non-transfected cells in the expression of bax protein. After the cells were affected by low-concentration ethanol. The killing rate, TUNEL index and sub-G1 apoptotic peek scale of the bax-transfected cells were all lower than those of the cells transfectcd with empty vector or non-transfected cells. Conclusions Overexprcssion of bax protein can enhance low-concentration ethanol-induced apoptosis in HCC-9204 hepatoma cells.