细胞复制性衰老及过氧化氢诱导的早衰过程中基因组DNA甲基化水平改变  

Changes of genome DNA methylation level during cellular replicative and premature senescence induced by hydrogen peroxide

作　　者：张文娟[1,2] 纪卫东[2] 杨淋清[2] 许玉玲[2] 张文姬[3] 庄志雄[2] 

机构地区：[1]南方医科大学公共卫生与热带医学学院毒理学系,广东广州510515 [2]深圳市疾病预防控制中心毒理研究室 [3]西南大学生物技术学院

出　　处：《毒理学杂志》2010年第1期1-5,共5页Journal of Toxicology

基　　金：国家自然科学基金重点项目(30571592;30630055;30700673);广东省自然科学基金博士启动项目(NO9451051501002539);深圳市科技计划重点资助项目(JH200505300503A);南方医科大学公共卫生与热带医学学院院长基金(NOGW200825)

摘　　要：目的检测体外培养的人胚肺成纤维细胞复制性衰老及过氧化氢诱导的细胞早衰过程中基因组DNA甲基化及甲基转移酶DNMT1的表达改变。方法应用5-甲基胞嘧啶免疫荧光法及[3H]甲基掺入法检测细胞衰老不同阶段细胞基因组DNA整体甲基化变化趋势,以Q-PCR和Western Blot方法检测DNMT1的mRNA和蛋白表达变化。结果细胞复制性衰老过程及早衰过程中,与年轻细胞组相比,5-甲基胞嘧啶免疫荧光强度逐渐降低,[3H]甲基掺入实验结果显示,甲基化的CpG%分别为年轻细胞组68.2%,中年细胞组41.9%,复制性衰老组16.1%,而早衰起始组63.5%,早衰持续组18.0%。DNMT1的mRNA和蛋白表达水平一致,在细胞复制性衰老过程中,DNMT1表达呈下降趋势,复制性衰老细胞组表达水平最低,而早衰起始组DNMT1表达显著升高,早衰持续组又降低至同复制性衰老组水平。结论本试验条件下,在细胞复制性衰老及早衰过程中,基因组DNA甲基化水平逐渐降低,基因组低甲基化是衰老细胞的伴随状态,与DNMT1水平降低有关。Objective Changes of genome DNA methylation and the expression of DNMT1 were observed during cellular replicative and premature senescence induced by hydrogen peroxide of human embryonic lung fibroblasts（HEFs）.Methods The genome DNA methylation levels were detected by 5-methylcytosine immunofluorescence assay and[3H]Methyl incorporation assay during different cell stages.The mRNA and protein expression for DNMT1 were measured by Real-time quantitative PCR and Western Blot.Results The mean density of fluorescence of 5-methylcytosine decreased gradually during cellular replicative and premature senescence,compared to young cells.An average proportion of methylated CpG was 68.2% in young cells,41.9% and 16.1% in mid-aged and replicative senescent cells separately,while 63.5% and 18.0% in premature senescence initiation and persistence status respectively according to the[3H]methyl incorporation assay.The alterations at protein level were similar to those at the mRNA level for DNMT1.DNMT1 decreased gradually with increased passages during cellular replicative senescence.However,the expression of DNMT1 increased in premature senescence initiation period and decreased in premature senescence persistence status as well as replicative senescence.Conclusion The genome methylation level decreased gradually during the premature as well as replicative senescence,which was associated with the reduction in the expression of DNMT1,reflecting global hypomethylation as a distinct feature of senescent cells.

关 键 词：细胞衰老 DNA甲基化 DNMT1 人胚肺成纤维细胞 

分 类 号：R994.6[医药卫生—毒理学]