细胞衰老过程中P16表观遗传学修饰研究  

Epigenetic Modifications of PI6 during Cellular Senescence

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

机构地区：[1]中山大学公共卫生学院预防医学系,广东广州510080 [2]深圳市疾病预防控制中心毒理研究室,广东深圳518020 [3]广州医学院化学致癌研究所,广东广州510182

出　　处：《环境与健康杂志》2008年第11期941-945,共5页Journal of Environment and Health

基　　金：国家自然科学基金重点资助项目(30571592,30630055,30700673);广东省自然科学基金资助项目(04002730);国家“973”计划基金资助项目(2002CB512903);深圳市科技计划重点资助项目(JH200505 300503A)

摘　　要：目的检测体外培养的人胚肺成纤维细胞复制性衰老过程及细胞早衰阶段中P16的表观遗传学调控作用。方法在细胞复制性衰老过程(同步培养的22 PDL正常人胚肺成纤维细胞于约50%融合度时进行400μmol/LH_2O_2处理,每天用H_2O_2染毒工作液染毒1次,每次2 h,持续4 d)中,将正常人胚肺成纤维细胞分为年轻细胞组(22 PDL)、中年细胞组(35 PDL)、复制性衰老细胞组(49 PDL);将经400μmol/L H_2O_2染毒4 d的22 PDL人胚肺成纤维细胞继续培养7 d,设为早衰细胞组。荧光定量PCR检测P16的mRNA表达水平,甲基化特异PCR检测其启动子区-846639 bp的甲基化水平,染色质免疫沉淀结合定量PCR检测其相应启动子区组蛋白修饰情况,包括组蛋白H3、H4乙酰化和H3(Lys4)及H4(Lys20)甲基化修饰。结果与年轻细胞组相比,中年细胞组P16的mRNA表达降低,复制性衰老细胞组和早衰细胞组P16的mRNA表达升高,差异均有统计学意义(P<0.05)。在P16启动子区,第一外显子上游-1000 bp内,其CpG岛片段长度为995 bp,甲基化特异性PCR(MSP)扩增片段位于CpG岛内-846～-639 bp之间,长度为208 bp。中年细胞组、复制性衰老细胞组、早衰细胞组甲基化引物扩增产物的相对含量分别为0.42、0.34、047,未甲基化引物扩增产物的相对含量分别为0.61、0.96、0.79。在P16 IP1启动子区(-685～-489 bp),复制性衰老细胞组及早衰细胞组以组蛋白H4(Lys20)甲基化修饰为主;在P16IP2启动子区(-229～-60 bp),复制性衰老细胞组受组蛋白H3、H4乙酰化和H4(Lys20)甲基化联合修饰,早衰细胞组以H3、H4乙酰化修饰为主。结论细胞衰老过程中,P16启动子区的组蛋白修饰联合调控其mRNA表达,复制性衰老与早衰的调控机制存在差异。Objective To understand the epigenetic regulations of P16 during cellular replicative senescence and premature senescence induced by hydrogen peroxide of human embryonic lung fibroblasts （HEFs）. Methods The normal HEFs were divided into young cells （22 PDL）, mid-aged cells （35 PDL） and replicative senescent cells （49 PDL） during replicative senescence. The synchronously cultured 22 PDL HEFs were exposed for 2 h to 400 umo]/L H202 at half confluence on daily basis. The procedure lasted for 4 consecutive days. And then the treated cells were cultured for another 7 days, called premature senescent cells. The mRNA level of P16 was detected by fluorescence quantitative PCR. The methylation level in the promoter region -846 ^-639 bp was observed by methylation-specific PCR （MSP）. The histone modifications was detected by chromatin immunoprecipitation-QPCR assay, including acetylation for H3, H4 and methylation for H3 （Lys4） and H4 （Lys20）. Results In the process of cellular senescence, the mRNA level of P16 decreased in mid-aged cells,but increased significantly in both replieative and premature senescent cells compared with that of young cells （P〈0.05）. In the - 1 000 bp of the first exon upstream promotor region of P16, the length of CpG island was 995 bp. The amplified fragment of MSP was 208 bp, located in -846--639 bp. The relative quantity was 0.42, 0.34 and 0.47 of methylated fragment for mid-aged cells, replicative senescent cells and premature senescent cells respectively, and 0.61, 0.96 and 0.79 of unmethylated fragment respectively. In the promoter （-685--489 bp） of P16, the main histone modifications was H4（Lys20） methylation for both replicative and premature senescence, while in the region （-229^-60 bp） was H3, H4 aeetylation and H4K20 methylation jointly in replicative senescence, and H3 and H4 acetylation in premature senescence. Conclusion The histone modifications in the promoter region take part in regulating the mRNA expression for P16 during cellular se

关 键 词：细胞衰老 基因 P16 甲基化 乙酰化 

分 类 号：R994.6[医药卫生—毒理学]