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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.
Objective To investigate the expression of wild-type p16 gene in hepatocellular carcinoma cells and its inhibitory effect on the growth of hepatocellular carcinoma cells, so as to provide a new way of studying the pathogenesis of hepatocellular carcinoma and to provide a theoretical basis for gene therapy of hepatocellular carcinoma.Methods Subcloning Type p16 gene and an unrelated DNA sequence were cloned into the eukaryotic expression vector pcDNA3 by double sticking and transfected into human hepatocellular carcinoma cell line 7721 by lipofectamine 2000. The expression of p16 gene mRNA was observed by in situ hybridization The expression of p16 gene was analyzed by laser scanning confocal microscope. The growth of 7721 cells was detected by MTT assay and flow cytometry (FCM), and the effect of p16 gene on the growth of hepatocellular carcinoma cell lines was analyzed.Results Endonuclease digestion Confirmed that wild-type wild-type p16 gene was cloned into eukaryotic expression vector, confirmed by in situ hybridization wild-type p16 gene expression in 7721 cells by confocal laser confocal analysis confirmed that transfected cells 7721 P16 The protein expression was significantly higher than that of the control group, and the cell cycle analysis showed that the cells in C_0 ~ G_1 phase were 29% ~ 41% .Conclusion Transfection of exogenous Green type p16 gene can be increased to reduce the expression level of p16 gene expression of P16 protein in liver cancer cells, thereby inhibiting p16 gene reduced the growth of cells.