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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P66Shc启动子区的组蛋白修饰变化。方法按传代情况将人胚肺成纤维细胞分为年轻细胞(22 population doubling levels,22PDL)组、中年细胞(35PDL)组、复制性衰老细胞(49PDL)组和氧化应激诱导的早衰细胞(premature senescence,PS)组。检测P66Shc的mRNA表达及其启动子区IP1、IP2组蛋白修饰,包括组蛋白H3、H4乙酰化和H3(Lys4)甲基化修饰。结果与年轻细胞组比较,中年细胞组、复制性衰老细胞组和早衰组人胚肺成纤维细胞P66Shc mRNA表达水平均升高,差异有统计学意义(P<0.05,P<0.01)。在P66Shc IP1启动子区(-1 641～-1 392 bp),中年细胞组和复制性衰老细胞组以H4乙酰化修饰为主,而早衰细胞组受H3、H4乙酰化和H3(Lys4)甲基化的联合修饰;在P66Shc IP2启动子区(-129～45 bp),中年细胞组和复制性衰老细胞组受H3(Lys4)甲基化修饰,而早衰细胞组受H4乙酰化和H4(Lys4)甲基化联合修饰。结论在细胞衰老过程中,P66Shc启动子区的组蛋白修饰参与其mRNA表达调控,复制性衰老与早衰的调控机制存在差异。
Objective To detect the histone modifications of the P66Shc promoter region during the replication of human embryo lung fibroblasts in vitro and the premature cell death induced by hydrogen peroxide. Methods Human embryonic lung fibroblasts were divided into 22 POPs (22 population doubling levels), middle-aged cells (35 PDL), replicative senescence cells (49 PDL) and oxidative stress-induced premature cells premature senescence, PS) group. The mRNA expression of P66Shc and its IP1 and IP2 histone modifications in promoter region were detected, including histone H3, H4 acetylation and H3 (Lys4) methylation modification. Results Compared with the young group, the expression of P66Shc mRNA in the human embryonic lung fibroblasts in the middle-aged group, the replication-senescent group and the premature aging group were significantly increased (P <0.05, P <0.01). In the P66Shc IP1 promoter region (-1 641 ~ -1 392 bp), the H4 acetylation was mainly expressed in the middle-aged and senescent cell groups, whereas the pre-aging cells were acetylated by H3 and H4 and H3 (Lys4) Methylation. In the P66Shc IP2 promoter region (-129 ~ 45 bp), the middle-aged cell group and the replicative senescent cell group were methylated by H3 (Lys4) methylation, while the premature cell group was H4 acetylated H4 (Lys4) methylation combined modification. Conclusions During the process of cellular senescence, the histone modification of P66Shc promoter is involved in the regulation of its mRNA expression. There are differences in the mechanisms of regulation between replicative senescence and premature senility.