5-氮胞苷对转化后间充质干细胞增殖、衰老及p16^(INK4a)表达的影响  

Effects of 5-Azacytidine on proliferation,senescence and p16^(INK4a) gene expression of transformed mesenchymal stem cells

作　　者：王玉竹 郑勇[1] 何柳[1] 万瑜[2] 宋健[1,2] 

机构地区：[1]武汉大学医学院人体解剖与组织胚胎学系 [2]武汉大学医学院结构生物学研究中心,武汉430071

出　　处：《中国组织化学与细胞化学杂志》2015年第1期9-14,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金资助(30971475);中央高校基本科研业务专项资金(2042014kf0060)

摘　　要：目的观察5-氮胞苷对体外自发转化的间充质干细胞增殖、衰老及p16INK4a基因表达的影响。方法采用细胞计数、SA-β-gal染色、Western Blot和重硫酸盐修饰后测序(BSP)检测体外自发转化的大鼠间充质干细胞在不同浓度5-氮胞苷处理后增殖、衰老、p16INK4a表达及p16INK4a基因启动子区CpG岛甲基化水平的变化。结果细胞计数显示5-氮胞苷可剂量依赖性地抑制转化后间充质干细胞的增殖,但增殖受抑的细胞SA-β-gal染色仍呈阴性。BSP及Western Blot分析显示:转化后间充质干细胞p16INK4a基因启动子区CpG岛呈现高水平甲基化(87.30±2.39%)。5-氮胞苷可在一定程度上降低该基因的甲基化,使p16INK4a表达得以部分恢复;但即使是高浓度高达100μmol/L,5-氮胞苷也只能使p16INK4a基因甲基化降低到46.20±1.65%。结论 5-氮胞苷可显著抑制转化后间充质干细胞的增殖,但并不能促使这些细胞重新恢复衰老状态;其原因是5-氮胞苷单独作用不足以完全解除p16INK4a基因启动子区DNA的高甲基化。Objective To observe the effects of 5-Azacytidine on proliferation,senescence and p16INK4 a gene expression of transformed mesenchymal stem cells(tMSCs).Methods Rat tMSCs were treated with different concentrations of 5-Azacytidine.Their proliferation,senescence,and p16INK4 agene expression and DNA methylation were analyzed with cell counting,SA-β-gal staining,Western Blot and bisulfite sequencing PCR(BSP),respectively.Results Cell counting showed that 5-Azacytidine significantly inhibited tMSC's proliferation in a dose-dependent manner,but SA-β-gal staining analysis demonstrated no restoration of the senescent phenotype in these 5-Azacytidine-treated cells.BSP and Western analysis showed that the CpG island in the promoter of the p16INK4 agene in tMSCs was highly methylated(87.30±2.39%).5-Azacytidine reduced the methylation level and partly restored the gene's expression.But even at a high concentration,such as 100μmol/L,5-Azacytidine only reduced the CpG island methylation to a level of 46.20±1.65%.Conclusion 5-Azacytidine inhibits tMSC's proliferation but its unable to restore the cell's senescence phenotype.This is because 5-Azacytidine alone is not enough to completely remove the inhibitory methyl modification of the p16INK4 agene.

关 键 词：间充质干细胞 细胞增殖 细胞衰老 5-氮胞苷 P16^INK4A基因 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]