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

doi:10.3864/j.issn.0578-1752.2013.02.018

摘要/Abstract

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

岳群华, 袁建龙, 郝斐, 靳木子, 王景园, 杨文亮, 刘东军, 仓明. 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[J]. 中国农业科学, 2013, 46(2): 385-393.

YUE Qun-Hua, YUAN Jian-Long, HAO Fei, JIN Mu-Zi, WANG Jing-Yuan, YANG Wen-Liang, LIU Dong-Jun, CANG Ming. Transfected FST Gene into Ovine Muscle-Drived Myogenic Cells to Regulate the Expression of MSTN Gene[J]. Scientia Agricultura Sinica, 2013, 46(2): 385-393.

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参考文献

[1]Wells D N, Misica P M, McMillian W H. Production of cloned bovine fetues following nuclear transfer with cells from a fetal fibroblast cell line. Theriogenology, 1999, 60: 996-1005.

[2]Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J Y, Doguchi H, Yasue H, Tsunoda Y. Eight calves cloned from somatic cells of a single adult. Science, 1998, 282(5396): 2089-2095.

[3]Baguisi A, Behboodi E, Melican D T, Pollock J S, Porter C A, Midura P, Palacios M J, Ayres S L, Denniston R S, Hayes M L, Destrempes M M, Cammuso C, Williams J L, Nims S D, Ziomek C A, Meade H M, Godke R A, Gavin W G, Overström E W, Echelard Y. Production of goats by somatic cell nuclear transfer. Nature Biotechnology, 1999, 17(5): 456-461.

[4]Polejaeva I A, Chen S H, Vaught T D, Page R L, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares D L, Colman A, Campbell K H. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 2000, 407(6800): 86-90.

[5]Shin T, Kraemer D, Pryor J, Ling L, Rugila J, Howe L, Buck S, Murphy K, Lyons L, Westhusin M. A cat cloned by nuclear transplantation. Nature, 2002, 41(6874): 859.

[6]Woods G L, White K L, Vanderwall D K, Li G P, Aston K I, Bunch T D, Meerdo L N, Pate B J. A mule cloned from fetal cells by nuclear transfer. Science, 2003, 301: 1063.

[7]Palmiter R D, Brinster R L, Hammer R E, Trumbauer M E, Rosenfeld M G, Birnberg N C, Evans R M. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature, 1992, 24: 429-433.

[8]张贺, 王承利, 王峰. 转基因研究进展. 动物医学进展, 2008, 29(7): 59-62.

Zhang H, Wang C L, Wang F. Progress on transgenic animals. Progress in Veterinary Medicine, 2008, 29(7): 59-62. (in Chinese)

[9]Kim T H, Barrera L O, Qu C, van Calcar S, Trinklein N D, Cooper S J, Luna R M, Glass C K, Rosenfeld M G, Myers R M, Ren B. Direct isolation and identification of promoters in the human genome. Genome Resource, 2005, 15(6): 830-839.

[10]Meng Q Y, Chen Z Q, Yu Z Q, Xie Q F, Li N. Increased body weight via injecting myogenic expression growth hormone releasing hormone(GHRH) plasmid DNA into sheep. Animal Biotechnology, 2004, 15(2): 175-192.

[11]Kemp P R, Osbourn J K, Grainger D J, Metcalfe J C. Cloning and analysis of the promoter region of the rat SM22 alpha gene. Biochemistry, 1995, 310(3): 1037-1043.

[12]Lu Q L, Bou-Gharios G, Partridge T A. Non-viral gene delivery in skeletal muscle: a protein factory. Gene Therapy, 2003, 10: 131-142.

[13]Li X, Eastman E M, Schwartz R J, Draghia-Akli R. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nature Biotechnology, 1999, 17(3): 241-245.

[14]Robertson D M, Klein R, De Vos F L, McLachlan R I, Wettenhall R E, Hearn M T, Burger H G, de Kretser D M. The isolation of polypeptides with FST suppressing activity from bovine follicular fluid which are structurally different to inhibin. Biochem Biophys Resources Commun, 1987, 149(2): 744-749.

[15]Ueno N, Ling N, Ying SY, Esch F, Shimasaki S, Guillemin R. Isolation and partial characterization of follistatin: a singlechain Mr 35000 monomeric protein that inhibits the release of follicle- stimulating hormone. Biochemistry, 1987, 84(23): 8282-8286.

[16]茆达干, 杨利国, 何晓红, 张志杰, 张红琳. 抑制素与乙肝表面抗原融合基因免疫对大鼠生殖能力的影响. 畜牧兽医学报, 2006, 37(11): 1160-1166.

Mao D G, Yang L G, He X H, Zhang Z J, Zhang H L. Effect of inhibin gene immunization fused with HbsAg on rat reproductive performance. Acta Veterinaria et Zootechnica Sinica, 2006, 37(11): 1160-1166. (in Chinese)

[17]张德坤, 杨利国, 张红琳, 王文, 乌娜尔汗, 纪平, 古武昌. 抑制素基因免疫诱导单胎绵羊孪生的研究. 中国农业大学学报, 2004, 9(4): 40-45.

Zhang D K, Yang L G, Zhang H L,Wang W, Wunaerhan,Ji P, Gu W C. Inhibin gene immunization to induce sheep twinning. Journal of China Agricultural University, 2004, 9(4): 40-45. (in Chinese)

[18]Trendelenburg A U, Meyer A, Rohner D, Boyle J, Hatakeyama S, Glass D J. Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. Cell Physiology, 2009, 296(6): 1258-1270.

[19]Alexandra C. McPherron and Se-Jin Lee. Suppression of body fat accumulation in myostatin-deficient mice.The Journal of Clinical Investigation, 2002 , 109(5): 595-601.

[20]Lee S J. Regulation of muscle mass by myostatin.PNAS, 2001, 98(16): 9306-9311.

[21]Mcpherron A C, Lawler A M, Lee S J. Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature, 1997, 387(6628): 83-90.

[22]王玮, 袁建龙, 金永, 朱兵, 岳群华, 梁浩, 郭旭东, 刘东军, 仓明. 构建骨骼肌特异表达人FST基因载体及其稳定转染绵羊胎儿成纤维细胞. 生物技术通报, 2011, 11: 140-145.

Wang W, Yuan J L, Jin Y, Zhu B, Yue Q H, Liang H, Guo X D, Liu D J, Cang M. Construction of a skeletal muscle specific expression vector of follistatin and its stable transfection into sheep fetal fibroblasts cells. Biotechnology Bulletin, 2011,11: 140-145. (in Chinese)

[23]焦泽华, 魏著英, 白春玲, 段彪, 程磊, 李光鹏. 大鼠肌肉卫星细胞的分离培养鉴定与诱导分化. 农业生物技术学报, 2011, 2: 302-307.

Jiao Z H, Wei Z Y, Bai C L, Duan B, Cheng L, Li G P. Isolation, identification and differentiation of rat muscle satellite cell. Journal of Agricultural Biotechnology, 2011, 2: 302-307. (in Chinese)

[24]Kocamis H, Gulmez N, Aslan S, Esch F, Shimasaki S, Guillemin R. Follistatin alters myostatin gene expression in C2C12 muscle cells. Acta Veterinaria Hungarica, 2004, 52(2): 135-141.

[25]Chang C H, Grant A L, Strasburg G M, Bergen W G, Merkel R A, Helferich W G. RNA transcripition in porcine skeletal muscle nuclei during postnatal development. Experimental Biology and Medicine, 1994, 206(2): 162-168.