重组肌肉抑制素功能分析及其对鸡肌肉发育的抑制作用  

Functional Characterization of Recombinant Myostatin and Its Inhibitory Role to Chicken Muscle Development

作　　者：杨威[1] 王琨[1] 陈岩[1] 张勇[1] 黄波[1] 朱大海[2] 

机构地区：[1]哈尔滨工业大学分子与细胞发育生物学实验室,哈尔滨150001 [2]中国协和医科大学基础医学院医学分子生物学国家重点实验室,北京100005

出　　处：《生物化学与生物物理学报》2003年第11期1016-1022,共7页

基　　金：国家高技术研究发展计划 ( 863计划 )资助项目 (No .2 0 0 1AA2 2 2 0 3 1) ;国家杰出青年基金资助项目 (No .3 0 0 2 5 0 )~~

摘　　要：肌肉抑制素 (myostatin)为TGF β超家族成员 ,具有典型的TGF β家族成员特有的分子结构与功能。为了深入阐明肌肉抑制素的作用机制 ,研究了重组肌肉抑制素蛋白对鸡胚成肌细胞和小鼠C2 C1 2 成肌细胞增殖与分化的作用 ,同时制备了肌肉抑制素特异性抗体。实验结果表明 ,重组肌肉抑制素对于成肌细胞的增殖过程具有极强的抑制作用 ,主要表现为抑制细胞周期由G1 期向S期的过渡 ,细胞生长速度显著变慢 ;同时重组肌肉抑制素也抑制成肌细胞分化为多核的融合肌管细胞 ,抑制肌肉分化标志myogenin和MHC的表达。由重组蛋白质制备的抗体能够特异性地识别人、小鼠、大鼠和鸡肌肉组织的内源肌肉抑制素。此外 ,通过免疫荧光技术还证实 。Myostatin is a recently discovered member of transforming growth factor β(TGFβ)superfamily and shares similar structure features with other members of TGFβ 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 G 1 to S phase and result in a cell cycle arrest at G 1. 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 endo genous 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 C 2C 12 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.

关 键 词：肌肉抑制素 细胞增殖 分化 抗体制备 细胞定位 鸡 肌肉发育 

分 类 号：Q51[生物学—生物化学]