摘要
Objective To investigate the effect of overexpression of bcl-2 on ethanol-induced apoptosis of primary hepatocellular carcinoma (HCC) cells.Methods The retrovirus expression vector pDOR-SB containing human bcl-2 cDNA was introduced into a human HCC cell line HCC-9204 by liposome-mediated transfection. pDOR-transfected and non-transfected HCC-9204 cells were used as controls. The expression of Bcl-2 protein by transfected HCC-9204 cells was detected by the immunohistochemical method. Then the cells were cloned with the limited dilution method continually until a monoclonal cell strain whose positive rate of Bcl-2 protein was 100% detected by flow cytometry was obtained. The killing rates of cells were detected by Methabenzthiazuron assay after the treatment of 6% ethanol for 6?h. The extent of apoptosis was analyzed by transferase-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry.Results Most of the pDOR-SB-transfected cells demonstrated Bcl-2 positive signals, while no signal was found in the controls. The positive rate of Bcl-2 protein detected by flow cytometry in the obtained monoclonal cell strain, which was named HCC-bcl2, was 100% after the cells had been cloned 3 times continually. The killing rate, TUNEL index and the scale of sub-G 1 apoptotic peak in HCC-bcl2 cells were all significantly lower than those in the control cells.Conclusion Overexpression of Bcl-2 protein suppresses ethanol-induced apoptosis of the HCC cell line HCC-9204.
目的 探讨bcl 2过表达对乙醇诱导的原发性肝细胞癌 (简称肝癌 )细胞凋亡的影响。方法 用脂质体介导的转染法将含有人bcl 2cDNA的逆转录病毒表达载体pDOR SB导入不表达bcl 2蛋白的人肝癌细胞系HCC 92 0 4中 ,并设转染pDOR和不转染的HCC 92 0 4细胞对照。用G 4 18筛选后获得转入bcl 2基因的细胞克隆 ,并用免疫细胞化学ABC法检测Bcl 2蛋白的表达情况。有限稀释法连续克隆化直至获得用流式细胞仪检测 10 0 %表达Bcl 2蛋白的单克隆细胞株。 6 %乙醇分别作用于上述细胞 6h后 ,甲噻唑蓝法检测细胞杀伤率 ,TUNEL染色法和流式细胞仪DNA含量检测分析细胞凋亡的发生程度。结果 转染pDOR SB的细胞大部分为Bcl 2蛋白阳性 ,而转染pDOR或未转染的HCC 92 0 4细胞中未见Bcl 2蛋白阳性信号。经过连续 3次克隆化后 ,流式细胞仪检测表明所获得的单克隆细胞株 10 0 %稳定表达Bcl 2蛋白 ,命名为HCC bcl2细胞。HCC bcl2细胞在 6 %乙醇作用 6h后的细胞杀伤率、TUNEL指数和亚二倍体凋亡峰比例均明显低于转染pDOR或未转染的HCC 92 0 4细胞。结论 Bcl 2蛋白过表达能够抑制乙醇诱导的肝癌细胞系HCC 92 0 4细胞凋亡。
