[Functional characterization of recombinant myostatin and its inhibitory role to chicken muscle development].

Wei Yang, Kun Wang, Yan Chen, Yong Zhang, Bo Huang, Da-Hai Zhu
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Abstract

Myostatin is a recently discovered member of transforming growth factor beta (TGFbeta) superfamily and shares similar structure features with other members of TGFbeta superfamily. For a better understanding of molecular mechanism of myostatin function, the production of C-terminal truncated form of recombinant myostatin protein (rMSTN) in E. coli was previously reported. Herein, the functional role of the recombinant myostatin in regulating myogenesis in a chicken embryonic myoblasts (CEMs) system was determined. By using flow cytometric analysis, the myostatin was found to inhibit cell cycle transition from G1 to S phase and result in a cell cycle arrest at G1. In addition, myostatin blocked the multi-nucleus myotube formation and caused a decreased expression of the muscle cell differentiation markers (myogenin and MHC) in CEMs. In this study, a rabbit polyclonal antibody against myostatin was produced and high affinity and specificity of this anti-myostatin antibody to recombinant and endogenous myostatin were assayed by Western blot analysis. Further studies showed that the antibody could also recognize the tissue endogenous myostatin of human, mouse and rat. A specific 40 kD band was detected in chicken muscle, which suggested that chicken myostatin might have different splicing pattern. Immunofluorescence assay indicated that myostatin predominantly existed in the cytosol in C2C12 cells. Taken together, the results show that myostatin inhibits chicken muscle cells proliferation and differentiation and down-regulates expression of two differentiation marker gene in CEMs. Remarkably, production of functional recombinant myostatin protein and its specific antibody provides important reagents for unraveling molecular mechanisms underlying myostatin action during myogenesis.

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重组肌生长抑制素的功能表征及其对鸡肌肉发育的抑制作用。
肌生长抑制素是最近发现的转化生长因子β (tgfβ)超家族成员,与tgfβ超家族的其他成员具有相似的结构特征。为了更好地了解肌生长抑制素功能的分子机制,以前有报道在大肠杆菌中产生c端截断形式的重组肌生长抑制素蛋白(rMSTN)。本研究确定了重组肌生长抑制素在鸡胚成肌细胞(CEMs)系统中调控肌肉发生的功能作用。通过流式细胞术分析,发现肌生长抑制素抑制细胞周期从G1期向S期转变,导致细胞周期阻滞在G1期。此外,肌生长抑制素阻断了多核肌管的形成,并导致CEMs中肌肉细胞分化标志物(肌生成素和MHC)的表达降低。本研究制备了兔肌生长抑制素多克隆抗体,并通过Western blot检测了该抗体对重组肌生长抑制素和内源性肌生长抑制素的高亲和力和特异性。进一步研究表明,该抗体还能识别人、小鼠和大鼠的组织内源性肌生成抑制素。在鸡肌肉中检测到一个特异性的40 kD条带,表明鸡肌肉生长抑制素可能具有不同的剪接模式。免疫荧光分析表明C2C12细胞的胞浆中主要存在肌生长抑制素。综上所述,肌生长抑制素抑制了鸡CEMs中肌细胞的增殖和分化,并下调了两种分化标志基因的表达。值得注意的是,功能性重组肌生长抑制素蛋白及其特异性抗体的产生为揭示肌生成过程中肌生长抑制素作用的分子机制提供了重要的试剂。
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