PTEN基因对人肝癌细胞系HHCC作用的研究

Effects of exogenous PTEN gene on human hepatocellular carcinoma cell line HHCC

目的 :研究PTEN基因对人肝癌细胞系HHCC细胞恶性表型及凋亡的影响。方法 :将PTEN真核表达载体 pBabe Puro PTEN及空质粒 pBabe Puro ,通过脂质体介导的基因转染方法 ,转入该基因表达缺失的HHCC细胞系中 ,嘌呤霉素筛选获得稳定表达PTEN基因的转染细胞。通过平板克隆形成试验、DNA凝胶电泳、相差显微镜和电镜 ,观察PTEN基因对人肝癌细胞系HHCC恶性表型及凋亡的影响。结果 :转染后的细胞 ,经原位杂交、免疫组织化学检测证实 ,有PTENmRNA及其蛋白的表达 ;平板克隆形成试验证实 ,转染PTEN基因后 ,细胞形成克隆的能力降低 ;转染PTEN的细胞在相差显微镜和电镜下可出现典型的凋亡形态学改变 ;DNA琼脂糖凝胶电泳呈现明显的梯状条带。结论 :外源性PTEN基因导入HHCC细胞后 ,可降低HHCC细胞的恶性表型 ,并促进凋亡发生。

Purpose To study the effects of exogenous PTEN gene on the malignant phenotype and apoptosis of human hepatocellular carcinoma(HCC) cell line HHCC. Methods The mammalian PTEN expressing recombinant, named pBabe Puro PTEN was transfected into HHCC cells by lipofectamine TM . After puromycin selection, HHCC cells which stably expressed PTEN were obtained. The effects of PTEN gene on malignant phenotype of HHCC cells were examined by the methods of colony formation on plate. Apoptosis was ascertained by morphological changes under phase contrast microscope and electronic microscope, DNA electrophoresis. Results Exogenous PTEN gene and its protein were successfully expressed in HHCC cells, which was confirmed by in situ hybridization and immunohistochemical staining. The ability to form colony decreased in HHCC cells transfected with pBabe Puro PTEN. Some classical morphological changes of apoptosis appeared under phase contrast microscope as well as electronic microscope. DNA ladder could also be demonstrated on DNA electrophoresis. Conclusion Exogenous PTEN suppresses the malignant phenotype and induces apoptosis of human hepatocellular carcinoma cell line HHCC.