中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (20): 3130-3137.doi: 10.3969/j.issn.2095-4344.3135

• 肌肉肌腱韧带组织构建 tissue construction of the muscle, tendon and ligament • 上一篇    下一篇

钟基因Bmal1、Clock参与调节骨骼肌的发育分化

杨新华1，闫银弟1，罗旭光2，杨艳萍1，李海荣1，崔慧林1，曹锡梅1   

  1. 山西医科大学，1组织学与胚胎学教研室，2微生物免疫学教研室，山西省太原市   030001
  • 收稿日期:2019-12-23 修回日期:2019-12-28 接受日期:2020-07-26 出版日期:2021-07-18 发布日期:2021-01-15
  • 通讯作者: 曹锡梅，副教授，硕士生导师，山西医科大学组织学与胚胎学教研室，山西省太原市 030001
  • 作者简介:杨新华，女，1993年生，山西省忻州市人，汉族，山西医科大学在读硕士，主要从事真核细胞基因调控机制的相关研究。
  • 基金资助:
    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

Bmal1 and Clock regulate the development and differentiation of skeletal muscle

Yang Xinhua1, Yan Yindi1, Luo Xuguang2, Yang Yanping1, Li Hairong1, Cui Huilin1, Cao Ximei1   

  1. 1Department of Histology and Embryology, 2Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Received:2019-12-23 Revised:2019-12-28 Accepted:2020-07-26 Online:2021-07-18 Published:2021-01-15
  • Contact: Cao Ximei, Associate professor, Master’s supervisor, Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • About author:Yang Xinhua, Master candidate, Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:
    the Natural Science Foundation of Shanxi Province, No. 201901D111185 (to CXM); the Natural Science Foundation of Shanxi Province for the Youth, No. 2014021028-1 (to CXM); the Science and Technology Innovation Foundation of Shanxi Medical University, No. 01201401 (to CXM

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摘要：

文题释义：
钟基因：钟基因是产生和维持机体昼夜节律的分子基础，包括Bmal1、Clock、Per、Cry、Rev-erbα和RORα等，参与调控睡眠-觉醒、激素水平、代谢等诸多生理活动，维持机体正常生理功能和稳态。钟基因的发现和对这些基因产物工作原理的揭示对了解生命及生命的运行原理，特别是对基因、行为和环境之间的关系有着非常重要的理论意义。三位美国科学家Jeffrey C. Hall、Michael Rosbash和Michael W. Young发现了决定生物钟行为的基因和这些基因产物的工作原理，获得2017年诺贝尔生理或医学奖。
骨骼肌发育分化：哺乳动物骨骼肌发育分化是一个复杂的生物学过程。胚胎发育早期，一部分具有成肌潜能的干细胞在不同基因的精细调控下激活、增殖，分化融合成肌管，进而形成有功能的骨骼肌。在此过程中，肌调节因子，如Myod、Myogenin等和众多转录因子协同作用，发挥重要的调控作用。

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立，有利于骨骼肌和其他组织器官的发育。
https://orcid.org/0000-0002-1149-9738 (杨新华)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 骨骼肌, 胚胎, 钟基因, 小鼠, C2C12成肌细胞

Abstract: 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 C2C12 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 C2C12 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-erbα were 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 C2C12 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-erbα were very weakly expressed. The expressions of MyoD and myogenin were gradually increased, specially myogenin. This indicates that C2C12 myoblasts are the optimal model for research on myogenic differentiation. Western blot analysis revealed that the protein levels of Bmal1 and Clock were highly expressed in C2C12 myoblasts, which might be necessary for remaining the activity of myogenic regulatory factor. MyoD was highly expressed, but the band of MyoD  did not achieve statistical significance. By the luciferase reporter assay, we found that Bmal1, Clock and MyoD  plasmids worked in synergistic fashion to regulate the expression of TCAP (Titin-cap). To conclude, Bmal1, Clock and MyoD are all key regulators of myogenic differentiation. Highly expressed MyoD may be related to phase separation and play an important role in the development and differentiation of skeletal muscle. Persistently expressed Bmal1 is beneficial to maintain the activity of muscle regulators. Lowly expressed Rev-erbα is beneficial to skeletal muscle maturation. Per1, which is consistently highly expressed during embryonic development, may be involved in the establishment of the inhibition of cyclic rhythms and is beneficial to the development of the skeletal muscle and other tissues and organs.

Key words: skeletal muscle, embryos, circadian Clock gene, mouse, C2C12

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