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目的检测体外培养的人胚肺成纤维细胞复制性衰老过程中及过氧化氢诱导细胞早衰阶段P53的表观遗传学修饰。方法荧光定量采用聚合酶链式反应法(PCR)检测P53的mRNA表达,甲基化特异PCR检测启动子区甲基化改变情况,染色质免疫沉淀结合定量PCR检测其组蛋白修饰,包括组蛋白H3,H4乙酰化和H3(Lys4)及H4(Lys20)甲基化修饰。结果细胞复制性衰老过程中,P53的mRNA表达在中年细胞组显著升高,复制性衰老细胞组也升高,早衰组无明显改变;细胞复制性衰老过程中,其启动子区-820 bp~-656 bp甲基化水平随增龄而降低,早衰组降低明显;复制性衰老细胞组及早衰组的组蛋白修饰在启动子区-889 bp~-676 bp以组蛋白H3(Lys4)甲基化修饰为主;-199 bp~-30 bp的修饰,复制性衰老细胞组以组蛋白H3,H4乙酰化修饰为主,早衰组以H4乙酰化,H3K4甲基化修饰为主。结论细胞衰老过程中,P53启动子区组蛋白修饰参与其mRNA表达调控,复制性衰老与早衰存在调控机制的差异。
Objective To detect the epigenetic modification of P53 in human embryo lung fibroblasts cultured in vitro and hydrogen peroxide-induced cell premature senescence. Methods The mRNA expression of P53 was detected by polymerase chain reaction (PCR). The methylation-specific PCR was used to detect the methylation of promoter region. Chromatin immunoprecipitation combined with quantitative PCR was used to detect the histone modifications including histone H3, H4 acetylation and H3 (Lys4) and H4 (Lys20) methylation modifications. Results During the process of cell-induced senescence, the mRNA expression of P53 was significantly increased in the middle-aged group and the senescent cell group was also increased, while in the premature senescence group, the mRNA expression of P53 was not significantly changed. In the process of cellular senescence, the promoter region -820 bp The methylation level of ~ -656 bp decreased with aging and decreased significantly in premature senility group. The histone modifications in the replication-senescent and premature senescent groups ranged from -889 bp to -676 bp in the promoter region to histone H3 (Lys4) The main modifications were -199 bp to -30 bp. The replicative senescent cells were mainly acetylated by histone H3 and H4. Premature senescence was mainly acetylated by H4 acetylation and methylated by H3K4. Conclusions During the process of cell senescence, the histone modification of P53 promoter is involved in the regulation of its mRNA expression, and there are differences in the regulatory mechanisms between replication-induced senescence and premature aging.