| Musclin is a protein which is specifically secreted by skeletal muscle.The previous study has shown that the expression of musclin gene is influenced by a variety of factors in the body,and the physiological function of musclin is related to the metabolism of sugar and fat.At present,the reports about musclin gene are very few,and the effect and mechanism of musclin gene on the cell for proliferation,the glucose uptake and the amino acid degradation of the undifferentiated myoblast have not been reported.Therefore,this study was designed to clarify these questions using the over-expression and RNA interference technology.lt will lay the foundation for revealing the role and mechanism of musclin gene in the genesis and development process of muscle,and provide the basis for the health of livestock and poultry and human.The first experiment:This experiment was divided into seven groups,including Untreated control, pcDNA vector,pcDNA-musclin,Control siRNA,Musclin siRNA,pcDNA vector+Control siRNA,pcDNA-musclin +Musclin siRNA.The cells of seven groups were treated by transient transfection respectively with different plasmid or siRNA,and the culture media and cells were collected.Then we did the relevant detections.The results showed:(1) The results of real-time fluorescence quantitative PCR for musclin showed:compared with the Untreated control group,the musclin mRNA expression levels of the pcDNA vecter group,the Control siRNA group and the pcDNA vecter+Control siRNA group had no significant change.Compared with the pcDNA vecter group.the musclin mRNA expression level of the pcDNA-musclin group increased by 2776.25 times,and it had extremely significant difference (P<0.01);Compared with the Control siRNA group,the musclin mRNA expression level of the Musclin siRNA group reduced by 99.29%,and it had extremely significant difference (P<0.01);Compared with the pcDNA vector+Control siRNA group,the musclin mRNA expression level of the pcDNA-musclin+Musclin siRNA group increased by 78.31 times, and it had significant difference (P<0.05).The above results suggested that we successfully achieved the efficient over-expression,the silence and the moderate over-expression of musclin gene.(2) The results of cell proliferation experiment showed:compared with the Untreated control group, the number of cells in the pcDNA vecter group,the Control siRNA group and the pcDNA vecter+Control siRNA group reduced,and that may be because the transfection reagent had a certain extent damage for cells.The number of cells in the pcDNA-musclin group had no significant difference with that in the pcDNA vecter group.Compared with the Control siRNA group,the number of cells in the Musclin siRNA group reduced by 54.48%,and it had extremely significant difference (P<0.01);Compared with the pcDNA vector+Control siRNA group,the number of cells in the pcDNA-musclin+Musclin siRNA group tended to decrease,but it had no significant difference.The above results suggested that the normal physiological level expression of musclin gene could promote the myoblasts proliferation.(3) The testing results of the nutrition indexes showed:①Compared with the Untreated control group.the glucose content of the cell culture fluid in the pcDNA vecter group,the Control siRNA group and the pcDNA vecter+Control siRNA group had no significant changes.Compared with the pcDNA vecter group.the glucose content of the cell culture fluid in the pcDNA-musclin group increased by 48.83%,and it had extremely significant difference (P<0.01);Compared with the Control siRNA group.the glucose content of the cell culture fluid in the Musclin siRNA group reduced by 28.07%,and it had significant difference (P<0.05);Compared with the pcDNA vector+Control siRNA group,the glucose content of the cell culture fluid in the pcDNA-musclin+Musclin siRNA group increased by 31.18%,and it had extremely significant difference (P<0.01).The above results suggested that the normal physiological level expression and the over-expression of musclin gene might inhibit the glucose uptake or promote the glycogen decomposition of myoblasts.②Compared with the Untreated control group,the urea nitrogen content of the cell culture fluid in the pcDNA vecter group,the Control siRNA group and the pcDNA vecter+Control siRNA group had no significant changes;Compared with the pcDNA vecter group.the urea nitrogen content of the cell culture fluid in the pcDNA-musclin group increased by 20.09%,and it had significant difference (P＜0.05); Compared with the Control siRNA group,the urea nitrogen content of the cell culture fluid in the Musclin siRNA group reduced by 25.17%,and it had significant difference (P<0.05);Compared with the pcDNA vector+Control siRNA group,the urea nitrogen content of the cell culture fluid in the pcDNA-musclin+ Musclin siRNA group increased by 17.33%,and it had significant difference (P<0.05).The above results suggested that the normal physiological level and the over-expression level of musclin could promote the decomposition of amino acids in myoblasts.(4) The results of the real-time fluorescence quantitative PCR showed:the expression of AKT1, AKT2,PI3Kp110,PPARy and GLUT4 in different groups had no obvious difference.The results of the real-time fluorescence quantitative PCR for LXRa showed:Compared with the Untreated control group, the expression of LXRa in the pcDNA vecter group,the Control siRNA group and the pcDNA vecter+Control siRNA group had no significant changes;Compared with the pcDNA vecter group, the expression of LXR a in the pcDNA-musclin group reduced by 41.24%,and it had extremely significant difference (P<0.01);Compared with the Control siRNA group.the expression of LXRa in the Musclin siRNA group reduced by 96.97%,and it had extremely significant difference(P<0.01);Compared with the pcDNA vector+Control siRNA group,the expression of LXRa in the pcDNA-musclin+Musclin siRNA group reduced by 6.06%,and it had significant difference(P<0.05).The above results suggested that the normal physiology level expression of musclin gene promoted the expression of LXRa,but the over-expression of musclin gene inhibited the expression of LXRa in myoblasts.The second experiment:In order to further research the functional mechanism of sheep musclin gene in skeletal muscle,we isolated the primary skeletal muscle satellite cell in sheep.Then we purified them by using differential attachment technique.The muscle satellite cell was identified by three methods,including the PCR amplification of myostatin gene.the cellular immunochemistry of desmin and the cell differentiate-on.The results showed that the isolated cells were sheep muscle satellite cells, and it laid a foundation for further research. |
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