猪cripto促进C2C12细胞分化  

Porcine cripto promotes the differentiation of C2C12 cells

作　　者：樊爽爽 张雪梅[1] 郭珍珍[1] 曾磊[1] 鲁绍芳 鲁维飞[1] 韩立强[1] 褚贝贝[1] 杨国宇[1] 王江[1] FAN Shuang-shuang;ZHANG Xue-mei;GUO Zhen-zhen;ZENG Lei;LU Shao-fang;LU Wei-fei;HANLi-qiang;CHU Bei-bei;YANG Guo-yu;WANG Jiang(Key Lab of Regulation on Animal Growth and Development of Agricultural Ministry of China, Henan Agricultural University, Zhengzhou 450002, Chin)

机构地区：[1]河南农业大学农业部动物生化与营养重点开放实验室

出　　处：《中国兽医学报》2018年第4期802-808,共7页Chinese Journal of Veterinary Science

基　　金：农业部“948”重点计划基金资助项目(2011-G35);农业部转基因重大专项基金资助项目(2014ZX0801015B)

摘　　要：应用分子生物学方法、荧光显微镜和实时荧光定量PCR技术，对cripto基因进行克隆、真核表达载体的构建，探讨cripto对C2C12细胞生长增殖的影响和肌细胞分化相关基因MyoD、MyoG、P21、MHC的表达变化。结果显示，phiC31cripto真核表达载体构建成功，荧光显微镜下可见转染phiC31载体和（ripto基因共转染的C2C12细胞表达强烈的绿色荧光，且对细胞的生长增殖无显著性影响；Q_PCR检测MyoD、MyoG、P21、MHC结果证实，猪cripto可以促进细胞分化因子MyoD、MyoG、P21、MHC的表达。结果表明，本试验成功地构建了C2C12-phiC31fripto过表达细胞系，Q—PCR结果分析发现cripto基因可以促进肌肉分化，为进一步探索cripto基因在肌肉分化中的分子机制提供理论依据。During the growth and development of skeletal muscle,the myostatin（MSTN） is an im- portant cytokine involved in the negative regulation of muscle growth. The cripto gene can partici- pate in skeletal muscle development regulation by regulating the activity of myostatin. In order to further study the effects of porcine cripto on the differentiation of C2C12 cells,This experiment was based on the successful construction C2C12-phiC31-cripto over-expression cell lines, which laid the foundation for the study of the myoblast differentiation. Application of molecular biology, fluorescence microscopy and real-time fluorescent quantitative PCR methods, tested gene cloning and construction of eukaryotic expression vector of cripto gene,besides we studied the effect of the stable expression of cripto on the cells proliferation,and the expression changes of MyoD, MyoG, P21and MHC in muscle cell differentiation. The eukaryotic expression vector phiC31-cripto was successfully constructed. The strong green fluorescence expression vector phiC31 and cripto gene were transfected into C2C12 cells under the fluorescence microscope;CCK-8 analysis results con- firmed that phiC31-cripto overexpression in the transfected cells had positive influence on cells proliferation;finally through fluorescence quantitative detection of MyoD, MyoG,P21, MHC con- firmed that the cripto gene could promote the differentiation of C2C12 cells. This experiment suc- cessfully constructed C2C12-phiC31-cripto cell lines, which provides the cellular models for the further exploring the synergistic effect of cripto in the process of muscle differentiation. The results showed that the cripto gene could promote muscle differentiation, which provided theoretical basis for further elucidation of the molecular mechanism of cripto gene in muscle differentiation.

关 键 词：肌肉生长抑制(MSTN)素 猪cripto基因 基因克隆 载体构建 C2C12细胞 真核表达 细胞分化 

分 类 号：Q78[生物学—分子生物学] S828[农业科学—畜牧学]