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目的 观察人DNAMTase反义基因转染后肝癌细胞系SMMC 772 1E 钙黏附蛋白表达的变化。方法 用基因重组技术构建包含人DNAMTase正、反义基因片段的真核表达载体 ;采用脂质体法将其转染入肝癌细胞系SMMC 772 1;采用RT PCR法观察DNAMTase基因mRNA、E 钙黏附蛋白基因mRNA表达变化 ;以甲基化特异的PCR法检测E 钙黏附蛋白基因启动子区域甲基化状态的改变 ;以免疫组化和流式细胞法检测E 钙黏附蛋白表达变化。结果 正、反义真核表达载体构建成功并转染入SMMC 772 1细胞 ,其中DNAMTase基因mRNA表达减低 ,E 钙黏附蛋白基因mRNA表达上调 ,E 钙黏附蛋白基因启动子区域呈现去甲基化状态 ,E 钙黏附蛋白表达上调。结论 抑制DNAMTase基因的表达可使肝癌SMMC 772 1细胞中E 钙黏附蛋白基因启动子区域去甲基化和表达增加 ;DNAMTase通过诱导启动子区域高甲基化 ,可使E 钙黏附蛋白基因低表达
Objective To observe the changes of the expression of cadherin in hepatocellular carcinoma cell line SMMC 772 1E after DNAMTase antisense gene transfection. Methods The eukaryotic expression vector containing positive and anti-sense DNA fragments of human DNAMTase was constructed by gene recombination technique. The recombinant plasmid was transfected into hepatocellular carcinoma cell line SMMC 772 1 by lipofectamine. The mRNA and protein expression of DNAMTase, The methylation status of E-cadherin gene promoter region was detected by methylation-specific PCR. The expression of E-cadherin protein was detected by immunohistochemistry and flow cytometry. Results The antisense eukaryotic expression vector was successfully constructed and transfected into SMMC 772 1 cells. The mRNA expression of DNAMTase gene was decreased, the expression of E-cadherin gene mRNA was up-regulated, and the promoter region of E-cadherin gene was demethylated , E-cadherin expression was up-regulated. Conclusion Inhibition of DNAMTase gene expression can demethylate and increase the promoter region of E-cadherin gene in hepatocellular carcinoma SMMC-772 1 cells. DNAMTase can induce low expression of E-cadherin gene by inducing promoter hypermethylation