基础医学与临床 ›› 2011, Vol. 31 ›› Issue (2): 187-191.

• 研究论文 • 上一篇    下一篇

siRNA阻断鼠成肌细胞myostatin表达对细胞增殖及分化能力的影响

孙顺昌1,彭运生1,贺敬波1,林志坚2   

  1. 1. 深圳市宝安区人民医院
    2. 北京大学深圳医院
  • 收稿日期:2010-06-03 修回日期:2010-07-28 出版日期:2011-02-05 发布日期:2011-03-14
  • 通讯作者: 孙顺昌 E-mail:shunchangsun@yahoo.com.cn

Effect of myostatin silence mediated by siRNA on cell proliferation and differentiation in myoblasts

  1. 1. People’s Hospital of Baoan, Shenzhen
    2.
  • Received:2010-06-03 Revised:2010-07-28 Online:2011-02-05 Published:2011-03-14
  • Contact: SUN Shun-chang E-mail:shunchangsun@yahoo.com.cn

摘要: 目的 研究myostatin表达被阻断后鼠成肌细胞的增殖及分化能力。方法 构建可阻断myostatin表达的siRNA表达载体,并转染鼠成肌细胞,用实时荧光定量RT-PCR和Western印迹鉴定myostatin表达。培养myostatin表达被阻断的成肌细胞,计数不同时间的细胞数和测定细胞表达的肌酸激酶。使用诱导分化培养液培养myostatin表达被阻断的成肌细胞,观察其分化成肌管的能力。结果 siRNA表达载体对成肌细胞myostatin表达的干扰率为81.6%,其干扰效果也被Western印迹所证实。Myostatin表达被阻断的成肌细胞数目增加(P<0.05),细胞内肌酸激酶活力升高(P<0.05)。正常成肌细胞培养7天可分化成肌管,myostatin表达被阻断后需10天才分化成肌管。结论 siRNA表达载体阻断成肌细胞的myostatin表达后细胞的增殖能力增强,分化被延迟,或许可成为治疗肌肉萎缩的一种新途径。

Abstract: Objective To study cell proliferation and differentiation of myoblasts with the myostatin silenced. Methods siRNA expression vector targeting the myostatin gene was constructed, then siRNA expression vector was transfected into myoblasts. The expression of myostatin in the transfected myoblasts was determined by real-time quantitative RT-PCR and Western blot. Transfected myoblasts were seeded in dishes, harvested at the indicated times, and counted. Creatine kinase was measured for transfected myoblasts. Transfected myoblasts were shifted to differentiation medium containing low fetal bovine serum, myotube formation was observated at the indicated times. Results Real-time quantitative RT-PCR showed that the rate of silencing of myostatin in transfected myoblasts was 81.6% for the siRNA expression vector, it was also testified by Western blot. The number of myoblasts transfected with the siRNA expression vector increased, and creatine kinase activity elevated than in control cells (P<0.05). After 7 days of incubation in differentiation medium, control myoblasts showed formation of myotubes. By contrast, formation of myotubes was observated after 10 days of incubation in differentiation medium for transfected myoblasts under the same conditions. Conclusion Cell proliferation capacity of myoblasts increases, and differentiation is inhibited when the myostatin is silenced with siRNA. Considering the above findings, it is likely that it is an alternative therapy method for muscle atrophy by silencing the myostatin gene with the siRNA expression vector.