绵羊Musclin基因真核表达载体的构建及其对肌细胞糖代谢和胰岛素作用的影响

Construction of Eukaryotic Expression Vector of Sheep Musclin Gene and Its Effects on Glucose Metabolism and Insulin Function in Myocytes

为了研究绵羊Musclin对糖代谢和胰岛素作用的影响,本试验运用生物信息学软件对其结构特征进行预测,并构建绵羊Musclin基因真核表达载体。分4组对绵羊胎儿成肌细胞进行处理:空载体组(pcDNA3.1)、Musclin过表达组(pcDNA3.1-Musclin)、空载体+胰岛素组(pcDNA3.1+Insulin)、Musclin过表达+胰岛素组(pcDNA3.1-Musclin+Insulin)。然后分别在分化48和72 h时收集细胞,检测Musclin对正常或添加胰岛素培养的分化状态绵羊肌细胞糖代谢的影响,并利用荧光定量PCR检测相关基因表达的变化。酶切鉴定和测序结果表明,成功构建了表达载体pcDNA3.1-Musclin。生物信息学分析表明,绵羊Musclin蛋白含有一个信号肽,并在28氨基酸位点处存在一个酶切位点。亚细胞定位在胞外,属于分泌蛋白。其二级和三级结构以α螺旋和无规则卷曲为主。细胞试验表明,绵羊Musclin基因过表达明显减少了正常或胰岛素处理的绵羊肌细胞糖原含量并增加了培养液中葡萄糖含量。诱导分化72 h时,过表达Musclin抑制了Musclin过表达组和Musclin过表达+胰岛素组绵羊肌细胞中IRS1、AKT1、AKT2、GLUT1、GLUT4基因mRNA表达,并促进了GSK3βmRNA表达;而诱导分化48 h时,只有GLUT4基因的表达受到抑制。综上所述,绵羊Musclin基因过表达可以影响肌细胞糖代谢并减弱胰岛素的作用,二者相互协调以维持糖代谢稳态。其作用机制涉及到上述相关基因的表达变化,且与绵羊成肌细胞的分化状态有关。

To investigate the effects of sheep Musclin on glucose metabolism and insulin function,the structure properties of sheep Musclin protein were predicted by using various bioinformatics softwares and the eukaryotic expression vector of Musclin gene was constructed.The sheep fetal myoblasts were divided into 4 groups and treated as follows:mock-vector group(pcDNA3.1),Musclin-overexpressed group(pcDNA3.1-Musclin),mock-vector+insulin group(pcDNA3.1+Insulin)and Musclin-overexpressed+insulin group(pcDNA3.1-Musclin+Insulin).Then the cells were collected at 48 and 72 h after differentiation induction,and the effects of Musclin on glucose metabolism in normal or insulin-treated differentiating myoblasts were analyzed and the changes of the related gene expression were detected by real-time PCR.The restriction endonuclease digestion and sequencing results indicated that the expression vector(pcDNA3.1-Musclin)was successfully constructed.The results of bioinformatics analysis showed that the sheep Musclin protein contained a signal peptide and had a restriction site at the 28th amino acid.The subcellular localization showed that it was in the extracellular space,and belonged to the secretory protein.Its secondary and tertiary structure mainly included alpha helixes and random coils.The cellular assays showed that the overexpression of sheep Musclin gene significantly reduced the glycogen content in the normal or insulin-treated sheep differentiating myoblasts and increased the glucose content in medium.The overexpression of Musclin inhibited the expressions of IRS1,AKT1,AKT2,GLUT1,GLUT4 mRNA,and promoted the expression of GSK 3βmRNA in the Musclin-overexpressed group and Musclin-overexpressed+insulin group sheep myoblasts induced differentiation for 72 h,while only the expression of GLUT 4 mRNA was inhibited after cells were induced differentiation for 48 h.In conclusion,the overexpression of the sheep Musclin gene affected the glucose metabolism and attenuated the functions of insulin in sheep differentiating myoblasts,and they coordinated with each other to maintain the homeostasis of glucose metabolism.The functional mechanisms of sheep Musclin were involved in the expression changes of the above genes and were related to the differentiation state of sheep myoblasts.