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目的 探讨细胞周期抑制蛋白p2 7在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 p2 7蛋白的肝癌细胞株。方法 提取含有人 p2 7基因的真核细胞表达载体pcDNA3 p2 7,用限制性内切酶EcoRⅠ和NotⅠ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 ,分别将真核细胞表达载体pcDNA3 p2 7和 pcDNA3导入不表达 p2 7蛋白的人肝癌细胞系HHCC细胞中 ,通过G4 18筛选获得转入目的基因的阳性细胞克隆。用免疫组织化学染色法和Westernblot检测p2 7蛋白的表达 ,经多次用有限稀释法连续克隆化 ,直至获得 10 0 %稳定表达 p2 7蛋白的肝癌细胞株。结果 琼脂糖凝胶电泳表明 ,可见 5 97bp的 p2 7基因片段。免疫细胞化学染色和Westernblot检测表明 ,转染pcDNA3 p2 7的HHCC细胞大部分有p2 7蛋白的表达 ;而转染空载体pcDNA3或未转染的HHCC细胞 ,均未见 p2 7蛋白表达。经连续 3次克隆化后 ,转染 pcDNA3 p2 7的HHCC细胞 10 0 %表达p2 7蛋白。结论 成功地获得了稳定表达p2 7蛋白的肝癌细胞株 ,命名为HCC p2 7。
Objective To investigate the regulatory function and mechanism of cell cycle arrest protein p27 in hepatocellular carcinoma cell apoptosis and to establish a hepatocellular carcinoma cell line stably expressing p27 protein. Methods The eukaryotic expression vector pcDNA3 p2 7 containing human p27 gene was extracted and digested with restriction endonucleases EcoRI and NotI and identified by agarose gel electrophoresis. The eukaryotic expression vectors pcDNA3 p2 7 and pcDNA3 were respectively introduced into human hepatocellular carcinoma cell line HHCC cells which did not express p27 protein by liposome-mediated gene transfection method. The positive genes transfected into the target gene were obtained by G418 screening Cell cloning. The expression of p27 protein was detected by immunohistochemical staining and Western blotting. After several times of continuous cloning by limiting dilution, 100% hepatoma cell lines stably expressing p27 protein were obtained. Results agarose gel electrophoresis showed that 5 97bp p2 7 gene fragment was observed. Immunocytochemical staining and Western blot showed that the majority of HHCC cells transfected with pcDNA3 p2 7 had p27 expression, whereas no p27 expression was detected in transfected empty vector pcDNA3 or untransfected HHCC cells. After three consecutive clones, 10% of HHCC cells transfected with pcDNA3 p2 7 expressed p27 protein. Conclusion The hepatoma cell line stably expressing p27 protein was successfully obtained and named as HCC p27.