沉默Dnmt1基因表达诱导大鼠骨髓间充质干细胞向心肌样细胞分化  

Silencing of the Dnmt1 Gene Induces Differentiation of Rat Mesenchymal Stem Cells to Cardiac Muscle-like Cells

作　　者：蒋昌科[1] 龚放[1] 

机构地区：[1]重庆医科大学附属永川医院儿科,重庆402160

出　　处：《中国生物化学与分子生物学报》2015年第4期408-413,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：重庆市卫生局重点项目(No.2011-1-082)~~

摘　　要：骨髓间充质干细胞(MSCs)具有向心肌样细胞分化的潜能.本室前期研究发现,MSCs在体外经DNA甲基转移酶(Dnmt)抑制剂5-氮胞苷诱导可分化为心肌样细胞.本研究证明,沉默DNA甲基化转移酶1(Dnmt1)基因表达,可诱导大鼠MSCs向心肌样细胞分化.本文采用表达Dnmt1 siRNA慢病毒感染MSCs,沉默Dnmt1表达.DNA甲基化分析显示,随着沉默Dnmt1时间延长(7-28 d),Gata-4基因上游DNA调控序列的Cp G甲基化水平明显降低,而Gata-4 mRNA的转录水平明显上调,说明敲减Dnmt1表达导致Gata-4基因激活.蛋白质印迹和/或免疫细胞化学揭示,与对照组比较,心肌相关基因MHC和c Tn T表达上调,而骨髓干细胞标志物CD90和CD29随转染时间延长表达下调.同时,实时定量PCR显示,心肌早期发育调控基因Nkx2.5 mRNA水平与Gata-4 mRNA相同,随表达Dnmt1 siRNA的慢病毒感染而上调.上述结果提示,敲减Dnmt1可降低心肌发育调控基因Gata-4启动子Cp G岛的甲基化水平,上调Gata-4基因的表达,诱导骨髓间充质干细胞向心肌样分化.Mesenchymal stem cells（MSCs） have been shown to be the potential to differentiate to cardiac muscle-like cells.We have previously demonstrated that exposure of MSCs to the Dnmt inhibitors5-azacytidine（5-aza-CR） may induce the differentiation of MSCs to cardiac muscle-like cells.Herein,we demonstrated that silencing Dnmt1 expression induced differentiation of rat MSCs to cardiac musclelike cells.First,Dnmt1 siRNA expressing lentivirus was constructed and infected MSCs to silence Dnmt1 expression.Methylation PCR assay showed that after different time of infection,the methylation levels of the Cp G island in the Gata-4 upstream sequence were markedly declined,while the levels of Gata-4mRNA were up-regulated,indicating the activation of the Gata-4 gene under the condition of Dnmt1 knockdown.Western blotting and/or immunocytochemistry revealed that in comparison with the control,the levels of cardiac MHC and c Tn T proteins were up-regulated,whereas the CD90 and CD29 proteins as MSCs markers were down-regulated in Dnmt-1 silenced MSCs.Furthermore,real-time quantitative-PCR（RT-q PCR） showed that the early cardiac muscle regulatory gene Nkx2.5 as well as Gata-4 was upregulated.These data suggest that silencing of Dnmt1 may induce the expression of the Gata-4 gene by reducing the levels of the Cp G island methylation in its promoter,thereby to promote MSC differentiation to cardiac-muscle related cells.

关 键 词：骨髓间充质干细胞 心肌 分化 DNA甲基转移酶1 

分 类 号：R54[医药卫生—心血管疾病]