钟基因Bmal1、Clock参与调节骨骼肌的发育分化  

Bmal1 and Clock regulate the development and differentiation of skeletal muscle

作　　者：杨新华 闫银弟 罗旭光[2] 杨艳萍[1] 李海荣[1] 崔慧林[1] 曹锡梅[1] Yang Xinhua;Yan Yindi;Luo Xuguang;Yang Yanping;Li Hairong;Cui Huilin;Cao Ximei(Department of Histology and Embryology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China;Department of Microbiology and Immunology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西医科大学组织学与胚胎学教研室,山西省太原市030001 [2]山西医科大学微生物免疫学教研室,山西省太原市030001

出　　处：《中国组织工程研究》2021年第20期3130-3137,共8页Chinese Journal of Tissue Engineering Research

基　　金：山西省自然科学基金面上项目(201901D111185),项目负责人:曹锡梅;山西省自然科学基金青年基金(2014021028-1),项目负责人:曹锡梅;山西医科大学科技创新基金(01201401),项目负责人:曹锡梅。

摘　　要：背景:骨骼肌具有昼夜节律,但骨骼肌发育分化过程中钟基因的作用机制及与肌调节因子之间的相互关系尚未完全研究清楚。目的:观察核心钟基因在不同胎龄小鼠胚胎和C2C12成肌细胞诱导分化过程中的时空表达特点,分析Bmal1、Clock和Myod的关系。方法:定量RT-PCR分析不同胎龄小鼠(10-17 d的ICR小鼠)胚胎和C2C12成肌细胞诱导分化过程中5个核心钟基因Bmal1、Clock、Per1、Cry1、Rev-erbα及肌调节因子Myod、myogenin、Tcap、MAZ的表达规律;Western blot检测C2C12诱导分化48,96和144 h BMAl1、CloCk和MYod蛋白的表达变化;荧光素酶活性分析Bmal1、CloCk和MYod质粒共转染对TCAP报告质粒的激活情况。实验方案经山西医科大学动物实验伦理委员会批准。结果与结论:①小鼠胚胎发育过程,Bmal1、Clock、Per1的表达随胎龄增加而升高,尤以Per1显著;Rev-erbα和Cry1呈低水平表达;②随着小鼠胚胎发育成熟,Myod、myogenin的表达逐渐增强,myogenin增长更迅速;Tcap的表达与肌节发育相关;MAZ高水平持续增长,可能参与调节骨骼肌的发育成熟;③C2C12诱导分化,Bmal1和Clock同步波动性升高;Cry1小幅同步升高;Per1和Rev-erbα呈低水平波动;Myod、myogenin呈上升趋势,尤以myogenin显著,提示该诱导分化模型有效;④Western blot表明,C2C12诱导分化前后,BMAl1、CloCk持续高表达可能利于维持肌调节因子的活性;诱导分化前后,MYod均呈高表达,随诱导分化时间延长,持续高表达的MYod较诱导分化前无统计学差异;⑤质粒共转染表明,Bmal1、CloCk与MYod质粒协同激活TCAP荧光素酶活性;⑥结果表明,Bmal1、Clock和Myod均是成肌细胞诱导分化的关键调节因子,持续高表达的Myod可能与相分离相关,对骨骼肌的发育分化发挥重要作用;持续表达的Bmal1利于维持肌调节因子的活性。低水平的Rev-erbα利于骨骼肌成熟;胚胎发育过程中持续高表达的Per1可能参与抑制周期性节律的建�BACKGROUND:Skeletal muscle has a circadian rhythm,but the mechanism of circadian Clock genes during the development and differentiation of the skeletal muscle and the interrelationship with muscle regulators are not fully understood.OBJECTIVE:To observe the spatio-temporal expression characteristics of circadian Clock genes in embryos of different mouse embryonic ages and during the differentiation of C2 C12 myoblasts as well as to examine the relationship of Bmal1,Clock and MyoD in the skeletal muscle development.METHODS:RT-qPCR was used to examine Bmal1,Clock,Per1,Cry1,Rev-erbα,as well as myogenic regulatory factors MyoD,myogenin,Tcap and MAZ from the embryos of ICR mice at embryonic ages of 10-17 days and during the differentiation of C2C12 myoblasts.Western blot was used to investigate the expression of BMAl1,CloCk and MYoD at 48,96,and 144 hours after differentiation of C2 C12 myoblasts.Luciferase reporter assay was used to analyze the activation of TCAP reporter plasmids by co-transfection of Bmal1,CloCk and MYoD plasmids.An approval for this study was obtained from the Animal Experimental Ethics Committee of Shanxi Medical University.RESULTS AND CONCLUSION:(1)With the development of mouse embryo,the expression of Bmal1,Clock,and Per1 were increased,especially Per1.Cry1 and Rev-erbαwere weakly expressed.(2)The expression intensity of MyoD and myogenin were gradually increased.In addition,the level of Tcap expression was related to sarcomere development.The expression of MAZ maintained a higher level and sustained increased in embryos,which might be involved in the development and differentiation of the skeletal muscle.(3)With the induced differentiation of C2 C12 myoblasts,the expressions of Bmal1 and Clock were synchronously increased.At the same time,the expression of Cry1 was up-regulated gradually.Per1 and Rev-erbαwere very weakly expressed.The expressions of MyoD and myogenin were gradually increased,specially myogenin.This indicates that C2 C12 myoblasts are the optimal model for research on myogen

关 键 词：骨骼肌 胚胎 钟基因 小鼠 C2C12成肌细胞 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学]