生物钟基因Clock对小鼠诱导性多能干细胞分化的影响  

The influences of circadian gene Clockon differentiation of induced pluripotent stem cells in mouse

作　　者：赵冉[1] 杨羊[1] 徐晨[1] 陆超 孙宁[1] 钱睿哲[1] 

机构地区：[1]复旦大学基础医学院生理与病理生理学系,上海200032

出　　处：《复旦学报（医学版）》2016年第5期505-512,共8页Fudan University Journal of Medical Sciences

基　　金：国家基础科学人才培养基金(J1210041);国家自然科学基金(81570771)~~

摘　　要：目的研究生物钟基因Clock对小鼠诱导性多能干细胞(induced pluripotent stem cell,iPSC)非定向分化的作用。方法将Teto-FUW-OSKM、M2rtTA、PsPAX2和PMD2.G4种质粒共转染293FT细胞,收集病毒上清,转染小鼠胚胎成纤维细胞(mouse embryo fibroblast,MEF)获得小鼠iPSC,并通过检测多能性基因表达及其体外分化能力对小鼠iPSC进行鉴定;通过RT-PCR检测小鼠iPSC在非定向分化和拟胚体形成过程中生物钟基因的表达,以及生物钟基因Clock下调后,与野生型细胞相比小鼠iPSC多能性基因的表达和形态学改变。结果通过慢病毒感染MEF的方式获得了小鼠iPSC;小鼠iPSC在非定向分化过程中,生物钟基因Clock、Per2、Rev-erbα表达升高;生物钟基因Clock下调后,小鼠iPSC分化能力下降,多能性基因Sox2、Oct4、Klf4表达升高。结论生物钟基因Clock对小鼠iPSC的非定向分化起到重要作用。Objective To study the relationship between circadian gene Clockand induced pluripotent stem cell（iPSC）differentiation in mouse. Methods Teto-FUW-OSKM,M2 rtTA,PsPAX2 and PMD2.G plasmid were transfected into 293 FT cells.Then virus-containing supernatants were harvested to infect mouse embryo fibroblast（MEF）.The characteristics of mouse iPSC（miPSC）were analyzed by pluripotent marker expression levels and the differentiation capacity in vitro.Real-time PCR was used to detect the expression of circadian Clock gene during spontaneous differentiation and the formation of embryoid body.After reducing the Clock gene expression level of miPSC,the cell morphological changes and the expression of pluripotency genes in miPSC were compared with wild type cells. Results We established miPSC by lentivirus inducing.The expression levels of circadian Clockgene,Per2 and Rev-erbαincreased in miPSC spontaneous differentiation process.After knocking down Clock gene,the differentiation of miPSC ability decreased as well as the expression levels of pluripotencygenes increased,such as Sox2,Oct4 and Klf4. Conclusions The circadian Clockgenes had an important influence on miPSC spontaneous differentiation.

关 键 词：生物钟基因 诱导性多能干细胞 非定向分化 小鼠 

分 类 号：R363[医药卫生—病理学]