中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (32): 5999-6005.doi: 10.3969/j.issn.2095-4344.2012.32.021

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

肌肉特异性微小RNA和成肌调节因子在C2C12细胞成肌分化 过程中的表达

宋 佳，曾 缨，郑民缨，张 成   

  1. 中山大学附属第一医院神经科，广东省广州市 510080
  • 收稿日期:2012-05-09 修回日期:2012-06-10 出版日期:2012-08-05 发布日期:2012-08-05
  • 通讯作者: 曾缨，博士，副教授，硕士生导师，中山大学附属第一医院神经科，广东省广州市 510080 drzengying@21cn.com
  • 作者简介:宋佳★，女，1987年生，汉族，中山大学附属第一医院在读硕士，现就职于河南省人民医院神经科，主要从事肌肉疾病的研究。 songjiasuxin@sina.com

Temporal expression patterns of muscle-specific microRNAs and myogenic regulatory factors during C2C12 cell differentiation

Song Jia, Zeng Ying, Zheng Min-ying, Zhang Cheng   

  1. Department of Neurology, First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
  • Received:2012-05-09 Revised:2012-06-10 Online:2012-08-05 Published:2012-08-05
  • Contact: Zeng Ying, M.D., Associate professor, Master’s supervisor, Department of Neurology, First Affiliated Hospital of SunYat-sen University, Guangzhou 510080, Guangdong Province, China drzengying@21cn.com
  • About author:宋佳★，女，1987年生，汉族，中山大学附属第一医院在读硕士，现就职于河南省人民医院神经科，主要从事肌肉疾病的研究。 songjiasuxin@sina.com

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546

被引次数

2

摘要：

背景：骨骼肌细胞的增殖与分化是由多因素参与的受严格调控的复杂生物学过程，其中，微小RNA和成肌调节因子作为调控基因表达的重要分子在成肌分化过程中起着关键的调控作用。
目的：探讨微小RNA-1，133，206在C2C12细胞成肌分化过程中的表达变化及成肌调节因子的调控作用。
方法：用含体积分数2%马血清的高糖DMEM培养基诱导C2C12细胞体外成肌分化，分别诱导分化后0，1，2，3，4，6 d时提取细胞总RNA，用于real time RT-PCR检测。
结果与结论：倒置显微镜观察和免疫荧光染色显示诱导C2C12细胞分化后3 d开始有肌管和肌球蛋白重链阳性细胞形成，并随诱导时间延长而增多。Real time RT-PCR检测显示，C2C12细胞成肌分化过程中，微小RNA-1，133，206与成肌调节因子MyoD和myogenin mRNA的表达水平均先上升后又恢复至接近分化前水平，而pax7 mRNA的表达则无明显变化。提示肌肉特异性微小RNA及成肌调节因子可能对C2C12细胞的成肌分化发挥一定的调控作用。

关键词: 成肌细胞, 分化, 肌管, 微小RNA, 成肌调节因子

Abstract:

BACKGROUND: The proliferation and differentiation of skeletal muscle cells are strictly regulated by multiple factors. Recently, more and more studies have indicated that microRNAs and myogenic regulatory factors as gene regulators play important roles in myogenesis.
OBJECTIVE: To explore the temporal expression patterns of muscle-specific microRNAs (miR-1, miR-133, and miR-206) in C2C12 cells during differentiation and the regulatory function of myogenic factors.
METHODS: C2C12 myogenic differentiation was induced in DMEM containing 2% horse serum. Inverted microscope was used to observe the alteration of their appearance, and immunofluorescence for myosin heavy chain was utilized to identify the differentiated C2C12 cells. At 0, 1, 2, 3, 4, 6 days after differentiation, the cellular totaI RNA of differentiated C2C12 cells was extracted. After reverse transcription, real-time polymerase chain reaction was used to determine the expression levels of three muscle-specific microRNAs as well as three myogenic regulatory factors (MyoD, myogenin and pax7) in differentiated C2C12 cells．
RESULTS AND CONCLUSION: After C2C12 cells were induced for 3 days, myotubes and MHC-positive cells began to form. Later on, more and more myotubes and MHC-positive cells appear and peaked at 6 days. The expression levels of miR-1, miR-133, miR-206, MyoD and myogenin were up regulated at first during C2C12 differentiation, and then recovered close to the levels of pre-differentiation. However, pax7 was not altered. The expression levels of muscle-specific microRNAs, MyoD and myogenin during C2C12 myogenic differentiation alter so strikingly that they might have effects on C2C12 differentiation; MyoD and myogenin might be involved in the induction of muscle-specific microRNAs.

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