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目的探讨人胚肺成纤维细胞复制性衰老及过氧化氢诱导的早衰过程中P66Shc的表达改变及其启动子区域的甲基化水平变化。方法荧光定量PCR方法检测细胞衰老过程中的mRNA表达改变,甲基化特异性PCR(MSP)定性检测甲基化的变化情况,亚硫酸氢盐修饰基因组结合克隆测序定量检测启动子区域CpG岛甲基化水平。结果人胚肺成纤维细胞复制性衰老及过氧化氢诱导的早衰过程中,P66Shc的mRNA表达水平逐渐升高,中年细胞组及复制性衰老细胞组mRNA表达分别升高至1.78和1.73倍,而早衰组却显著增高,至7.16倍;年轻细胞组、复制性衰老细胞组及早衰组P66Shc启动子区域CpG岛呈高甲基化水平,分别为94%、90%和90%。结论人胚肺成纤维细胞复制性衰老过程中P66Shc的mRNA表达水平逐渐升高,其启动子区CpG岛呈高的甲基化水平,不参与该基因表达调控。
Objective To investigate the changes of P66Shc expression in human embryonic lung fibroblasts (COPD) and the premature aging induced by H2O2 and the changes of methylation level in promoter regions. Methods Fluorescence quantitative PCR was used to detect the mRNA expression changes during cell senescence. Methylation-specific PCR (MSP) was used to detect the changes of methylation. The bisulfite modified genome was cloned and sequenced to detect the CpG island A Basic level. Results During the replication of human embryonic lung fibroblasts and hydrogen peroxide-induced premature senility, the mRNA expression of P66Shc increased gradually, and the mRNA expressions of middle-aged cells and replicative senescent cells increased to 1.78 and 1.73 times respectively, While the premature decay group was significantly increased to 7.16 times. The CpG island in the P66Shc promoter region of the young cell group, the replicative senescent cell group and the premature decay group was highly methylated, which was 94%, 90% and 90% respectively. Conclusion The expression of P66Shc mRNA in human embryonic lung fibroblasts during the process of senescence increased gradually. The CpG island in promoter region showed a high level of methylation, which was not involved in the regulation of gene expression.