中国农业科学 ›› 2013, Vol. 46 ›› Issue (2): 385-393.doi: 10.3864/j.issn.0578-1752.2013.02.018

• 畜牧·资源昆虫 • 上一篇    下一篇

转卵泡抑制素基因对绵羊肌源性细胞中 肌肉抑制素基因的调控

 岳群华, 袁建龙, 郝斐, 靳木子, 王景园, 杨文亮, 刘东军, 仓明   

  1. 内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特 010021
  • 收稿日期:2012-03-16 出版日期:2013-01-15 发布日期:2012-07-10
  • 通讯作者: 通信作者仓明;E-mail:cangming1970@yahoo.com.cn
  • 作者简介:岳群华,E-mail:yuequnhua@126.com
  • 基金资助:

    国家转基因生物新品种培育重大专项(2013zx08008003-003)、内蒙古自然科学重点项目(20080404zd08)

Transfected FST Gene into Ovine Muscle-Drived Myogenic Cells to Regulate the Expression of MSTN Gene

 YUE  Qun-Hua, YUAN  Jian-Long, HAO  Fei, JIN  Mu-Zi, WANG  Jing-Yuan, YANG  Wen-Liang, LIU  Dong-Jun, CANG  Ming   

  1. Key Laboratory of Ministry of Education of China for Mammal Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010021
  • Received:2012-03-16 Online:2013-01-15 Published:2012-07-10

摘要: 【目的】构建骨骼肌特异表达目的基因卵泡抑制素(follistatin,FST)的真核表达载体pCFCDs-red和 pSPFCDs-red,分别转染到不同细胞系中,检测FST及下游基因在RNA和蛋白水平的表达情况,同时测定骨骼肌特异启动子SP和α-actin的启动效率,以研究FST的特异表达对肌肉发育的影响。【方法】利用脂质体分别将真核表达载体pCFCDs-red和pSPFCDs-red转染到绵羊胎儿成纤维细胞(SFFCs)、骨骼肌卫星细胞(SMSCs)和SMSCs诱导肌管中,获得转基因细胞;对获得细胞的生长状况、细胞周期、细胞形态大小以及目的基因和下游基因的表达情况,分别用流式细胞仪、实时定量PCR和western blotting等方法进行检测分析。【结果】①转基因细胞系的生长趋势与非转基因细胞相似,转基因SMSCs细胞增殖的速度高于转基因SFFCs细胞;②流式细胞仪分析表明,转基因细胞系转染后70%以上细胞均处于G0/G1期,保持旺盛的分裂能力、具有单一峰值、细胞的整倍性好、细胞电子体积显著增大;③实时定量PCR及western blotting检测结果表明,转基因细胞系中FST的RNA及蛋白水平相比非转基因细胞系均上调;④转基因SMSCs和肌管相比转基因SFFCs细胞的FST表达量高,转pSPFCDs-red转基因细胞中FST的表达水平比转pCFCDs-red细胞系高。【结论】骨骼肌特异性启动子SP及α-actin能够在SMSCs及肌管中有效启动FST目的基因的表达,且在肌管中具有较高的表达量,SP启动子的启动效率较高于α-actin。在肌源性细胞中,FST的上调可以引起MSTN蛋白水平的下调,FST对骨骼肌细胞的生长发育具有一定促进作用。

关键词: FST , 人工合成启动子SP , 骨骼肌特异表达启动子&alpha, -actin , 骨骼肌卫星细胞 , 肌管

Abstract: 【Objective】In this research, skeletal muscle specific expressed follistatin(FST)eukaryotic expression vectors, pCFCDs-red and pSPFCDs-red, were constructed, then respectively transfected into different cell lines. FST and downstream gene expression situation in RNA and protein levels was detected, and the simultaneous start efficiency of skeletal muscle-specific promoter of sub-SP and α-actin was assayed, so that to understand the effect of specific expression of FST on the development of muscle.【Method】Using liposomes, the eukaryotic expression vectors of pCFCDs-red and pSPFCDs-red were transfected respectively into sheep fetal fibroblast cells (SFFCs), skeletal muscle satellite cells (SMSCs) and SMSCs induced muscle tube, and the transgenic cells were analyzed on status of cell growth, cell cycle, cell size and expression of target gene and downstream gene respectively by flow cytometry, Real-Time quantitative PCR and western blotting. 【Result】The growth of transgenic cells basically comply with the trend of non-transgenic cells, and transgenic SMSCs cell had a higher proliferation rates than that genetically modified SFFC. Flow cytometry analysis showed that more than 70% of cells of transgenic cell lines were in G0/G1 phase, maintained a strong ability to divide and had a single peak ,a good aneuploidy and volume increased significantly. Real-time quantitative PCR and western blotting results showed that RNA and protein levels of FST in the transgenic cell lines were raised compared to non-transgenic cell lines. Transgenic SMSCs and myotubes compared to transgenic SFFCs cell had higher expression of FST. Transfected pSPFCDs-red transgenic cells had higher expression level of FST than transfected pCFCDs-red cells.【Conclusion】Skeletal muscle specific promoter SP and α-actin can effectively start FST target gene in the SMSCs and myotubes, and FST have a higher expression level in myotubes. Start efficiency of SP promoter is higher than that of α-actin. In myogenic cells, the increased FST can cause the reduction of MSTN proteins level. FST has a certain role in promoting growth and development of skeletal muscle cells. Key words: FST; synthetic promoter SP ;specific promoter α-actin; skeletal muscle satellite cell; myotubes

Key words: FST , synthetic promoter SP , specific promoter α-actin , skeletal muscle satellite cell , myotubes