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目的:建立检测肝癌胰岛素样生长因子II(IGF-II)基因启动子P3甲基化状态的方法。方法:培养三株肝癌细胞系(BEL7402、SMMC7721、HepG2),选取正常肝组织3例;提取基因组DNA,亚硫酸氢钠修饰,巢式聚合酶链反应(nestedPCR)加甲基化特异性PCR(MSP)检测IGF-II基因启动子P3的甲基化状态,PCR产物经克隆、测序验证。结果:三株肝癌细胞系IGF-II基因启动子P3去甲基化,正常肝组织IGF-II基因启动子P3甲基化,目的片段无突变,甲基化修饰完全。结论:巢式PCR加甲基化特异性PCR可准确检测IGF-II基因启动子P3的甲基化状态。
Objective: To establish a method to detect the methylation status of promoter P3 of insulin-like growth factor II (IGF-II) gene in hepatocellular carcinoma. Methods: Three hepatocellular carcinoma cell lines (BEL7402, SMMC7721 and HepG2) were cultured and three normal liver tissues were selected. Genomic DNA, sodium bisulfite modification, nested PCR and methylation-specific PCR MSP) to detect the methylation status of IGF-II gene promoter P3, and the PCR products were confirmed by cloning and sequencing. Results: The three hepatocellular carcinoma cell lines were demethylated by promoter P3 of IGF-II gene, P3 was methylated by IGF-II gene promoter in normal liver tissue, and the target fragment was absent. Conclusion: Nested PCR plus methylation-specific PCR can accurately detect the methylation status of IGF-II promoter P3.