稳定表达Bcl┐2蛋白的肝细胞癌细胞株的建立与鉴定.docVIP

稳定表达Bcl┐2蛋白的肝细胞癌细胞株的建立与鉴定 作者：杨连君，王文亮，司晓辉 【关键词】 基因 　　关键词 :基因，bcl-2;癌，肝细胞;转染;基因表达 　　摘 要: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筛选获得转入目的基因的阳性细胞克隆.免疫细胞化学法检测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次克隆化后转染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. 　　Keywords:genes，bcl-2;carcinoma，hepatocellular;transfec-tion;gene expression 　　Abstract:AIM A hepatocellular carcinoma（HCC）cell strain which can express Bcl-2protein stably was established to elucidate the regulating function and mechanism of anti-apoptosis gene bcl-2in the apoptotic process of HCC.METHODS The retrovirus eukaryotic expression vector pDOR-SB containing human bcl-2gene 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-9204cells，which do not express Bcl-2protein，by lipo-some-mediated gene transfection method.The cell clones into which pDOR-SB or pDOR had been introduced were obtained with G-418selection.The expression of Bcl-2protein was detected using immunocytochemical method.The pDOR-SB-tranfected cells were cloned several times continually by lim-ited dilution method until an HCC cell strain that can express Bcl-2protein at a100%positive rate was obtained.RE-SULTS Agarose gel electrophoresis demonstrated a1.9-kb bcl-2gene fragment after pDOR-SB being cut with EcoRⅠ.The immunocytochemical detection indicated that Bcl-2pro-tein was expressed in most of the pDOR-SB-transfected cells，but there was no Bcl-2protein signal in the pDOR-transfect-ed or non-transfected HCC-9204cells.The pDOR-SB-trans-fe