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目的检测人胚肺成纤维细胞复制性衰老及过氧化氢诱导细胞早衰过程中组蛋白整体乙酰化修饰的改变。方法应用细胞免疫荧光实验观察组蛋白乙酰化水平变化,并基于ELISA样反应方法检测组蛋白总体去乙酰化酶的活性变化,荧光定量PCR检测乙酰化酶mRNA表达,荧光定量PCR和Western印迹检测去乙酰化酶表达变化及曲古霉素A对相应酶表达的影响。结果在细胞复制性衰老及细胞早衰过程中,组蛋白H3和H4整体乙酰化水平逐渐下降;去乙酰化酶活性逐渐降低;与年轻细胞组相比,中年细胞与复制性衰老细胞组P300表达下降;中年细胞组PCAF稍升高,复制性衰老细胞组降低;早衰起始组P300和PCAF均升高,早衰持续组P300降低;中年细胞组HDAC1表达稍降低;复制性衰老细胞组HDAC2稍降低;而HDAC3均降低;早衰起始组HDAC1,HDAC3有不同程度升高;早衰持续组HDAC2,HDAC3降低显著,而HDAC1明显升高。曲古霉素A诱导P300,PCAF表达,而降低HDAC1,HDAC2和HDAC3表达。结论组蛋白H3和H4整体低乙酰化是衰老细胞的伴随状态;细胞复制性衰老与过氧化氢诱导的早衰内在调控机制存在差别。
Objective To detect the changes of histone acetylation in human embryonic lung fibroblasts and H2O2-induced cell premature aging. Methods The changes of histone acetylation level were observed by immunofluorescence assay. The changes of histone deacetylase activity were detected by ELISA. The expression of acetylase mRNA was detected by fluorescence quantitative PCR. The fluorescence quantitative PCR and Western blotting were used to detect Changes of acetylase expression and the effect of trichostatin A on the expression of corresponding enzymes. Results During the process of cell-induced senescence and premature senescence, the overall acetylation levels of histone H3 and H4 decreased gradually and the activity of sirtuin decreased gradually. Compared with the young group, the expression of P300 in middle-aged and senescent cells Decreased in the middle-aged group; PCAF increased slightly in the middle-aged group, and decreased in the senescent cell group; P300 and PCAF increased in the premature senility group and P300 decreased in the premature senility group; the expression of HDAC1 in the middle-aged group decreased slightly; Slightly decreased; while HDAC3 decreased; HDAC1, HDAC3 increased in different degree in premature senility onset group; HDAC2 and HDAC3 decreased significantly in premature senile persistence group and HDAC1 increased significantly. Trichostatin A induces P300, PCAF expression and decreases HDAC1, HDAC2 and HDAC3 expression. Conclusion The overall hypoacetylation of histone H3 and H4 is a companion state of senescent cells. The mechanism of cellular senescence and hydrogen peroxide-induced intrinsic regulation of premature aging is different.