共聚焦方法分析p16基因的表达及对人肝癌细胞系7721生长的作用

Effect of p16 gene expression in growth of human hepatic carcinoma cell line 7721 with confocal microscopic analysis

目的探讨野生型 p16基因在肝癌细胞中的表达以及对肝癌细胞生长的抑制作用,为肝癌发病机制的研究提供一种新的思路,为肝癌基因治疗提供理论基础.方法采用亚克隆技术,将野生型 p16基因全长及一段无关DNA 序列,通过双粘端克隆入真核表达载体 pcDNA3中,用lipofectamine 2000介导的方法转染7721细胞(人肝癌细胞系).通过原位杂交观察 p16基因 mRNA 在细胞中的表达,用激光共聚焦方法分析 p16基因的蛋白表达产物,MTT 及流式细胞仪检测7721的生长状态,分析 p16基因对肝癌细胞株生长的影响.结果用内切酶酶切分析证实外源野生型 p16基因克隆入真核表达载体中,经原位杂交证实外源野生型 p16基因可以在7721细胞中表达,通过激光共聚焦分析证实,转染后的肝癌细胞7721中 P16的蛋白的表达明显高于对照组,对细胞进行周期分析表明,处于 C_0～G_1期细胞为29%～41%.结论通过转染外源野生型 p16基因可提高 p16基因表达减低肝癌细胞中 P16蛋白的表达量,从而抑制 p16基因表达减低细胞的生长.

AIM To explore the effect of p16 gene on the growth inhibition of hepatic carcinoma cell line 7721, Pathogenesis of hepatic carcinoma so as to offer theoretical basis for gene therapy. METHODS Using subclone technic,the wild type p16 gene and irrelative DNA sequence were cloned into an eukaryotic express vector pcDNA3.Using Lipofectamine 2000,the recombinate plasmid pcDNA3-p16 was transfected into the 7721 human hepatic carcinoma cell line.mRNA of p16 in the 7721 cell was detected with in situ hybridization.Confocal microscopy was used to analyze p16 gene protein expression.Growth state of transfected 7721 cell was examined by MTT and FCM. RESULTS Using restriction enzyme analysis,p16 gene was cloned in vector pcDNA3.Expression of ex ogenons p16 gene in 7721 cells was identified by in situ hybridization and confocal microscope quantitively.By cell cycle analysis,the cell in a period G_0-G_1 was about 29%-41%.The growth rate of 7721 transfected with p16 gene was markedly suppressed. CONCLUSION Transfection of wild type p16 gene into p16 gene can decrease the P16 protein expression in hepatic cancer cells and suppress cell growth by arresting cell cycle at G_1 phase.