稳定表达Bcl-2蛋白的肝细胞癌细胞株的建立与鉴定

目的 为了探讨抗凋亡基因bcl-2在肝细胞癌（简称肝癌）细胞凋亡过程中的调节功能及其作用机制，建立稳定表达Bcl－2蛋白的肝癌细胞株。方法 提取含有人bcl-2基因的逆转录病毒真核表达载体pDOR－SB质粒，用限制性内切酶EcoRI(酶切后经琼脂糖凝胶电泳鉴定，用脂质体介导的基因转染法分别将pDOR－SB和pDOR质粒导入不表达Bcl－2蛋白的人肝癌细胞系HCC－9204细胞中，通过G－418筛选获得转入目的的基因的阳性细胞克隆，免疫细胞化学检测Bcl-2蛋白的表达情况，经多次 用有限稀释法连续克隆化直至获得100％稳定表达Bcl-2蛋白的肝癌细胞株。结果 琼脂糖凝胶电泳可见1．9kb的bcl－2基因片断，免疫细胞化学检测表明转染了pDOR-SB的HCC－9204细胞大部分有Bcl-2蛋白的表达，而转染了pDOR空载体或未转染的HCC－9204细胞均未见Bcl－2蛋白表达，经过连续3次克隆后转染pDOR－SB的HCC－9204细胞100％表达Bcl-2蛋白。结论 成功地获得了稳定表达Bcl－2蛋白的肝癌细胞株，命名为HCC－bcl2。

Establishment and identification of the hepatocellular carcinoma cell strain which expresses Bcl-2 protein stably

AIM　A hepatocellular carcinoma (HCC) cell strain which can express Bcl-2 protein stably was established to elucidate the regulating function and mechanism of anti-apoptosis gene bcl-2 in the apoptotic process of HCC. METHODS　The retrovirus eukaryotic expression vector pDOR-SB containing human bcl-2 gene was extracted and identified by agarose gel electrophoresis after being cut with restricted endonuclease EcoRⅠ. The pDOR-SB and empty vector pDOR were introduced into a human HCC cell line HCC-9204 cells, which do not express Bcl-2 protein, by liposome-mediated gene transfection method. The cell clones into which pDOR-SB or pDOR had been introduced were obtained with G-418 selection. The expression of Bcl-2 protein was detected using immunocytochemical method. The pDOR-SB-tranfected cells were cloned several times continually by limited dilution method until an HCC cell strain that can express Bcl-2 protein at a 100% positive rate was obtained. RESULTS　Agarose gel electrophoresis demonstrated a 1.9-kb bcl-2 gene fragment after pDOR-SB being cut with EcoRⅠ. The immunocytochemical detection indicated that Bcl-2 protein was expressed in most of the pDOR-SB-transfected cells, but there was no Bcl-2 protein signal in the pDOR-transfected or non-transfected HCC-9204 cells. The pDOR-SB-transfected HCC-9204 cells expressed Bcl-2 protein at a 100% positive rate after being cloned for 3 times continually. CONCLUSION　HCC cell strain that can express Bcl-2 protein stably has been established successfully, and it is named as HCC-bcl2.