摘要
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。
Aim To establish carcinoma cell strain stably expressing p27 protein and explore the regulatory function and mechanism of cell cycle inhibitory protein gene p27 in the apoptotic process of HCC cells. Methods The eukaryotic expression vector pcDNA3 p27 containing human p27 gene was extracted and was identified by agarose gel electrophoresis after being cut with restriction endonuclease Eco R Ⅰand Not Ⅰ.The pcDNA3 p27 and empty vector pCDNA3 were introduced into a human HHCC cells which did not express p27 protein by liposome mediated gene transfection method. The cell clones into which pcDNA3 p27 or pcDNA3 had been introduced were obtained with G418 screening. The expression of p27 protein was detected by immunocytochemical staining and Western blot. The cells transfected by pcDNA3 p27 were cloned several times continually by limited dilution method until whole HHCC cells could express p27 protein.Results The atlas of agarose gel electrophoresis demonstrated a p27 gene fragment of 597 bp after pcDNA3 p27 being cut with Eco R Ⅰ and Not Ⅰ The detection of immunocytochemical staining and Western blot indicated that p27 protein was expressed in most of the cells transfected by pcDNA3 p27, but there was no p27 protein signal in the pDNA3 transfected or non transfected HHCC cells. The whole HHCC cells transfected by pcDNA3 p27 expressed p27 protein after cloning continually for 3 times.Conclusion HHCC cells expressing stably p27 protein has been established successfully and it is named HCC p27.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2002年第3期228-230,共3页
Chinese Journal of Cellular and Molecular Immunology
关键词
P27
肝细胞肝癌
转染
基因表达
p27
hepatocellular carcinoma
transfection
gene expression
