The Role Of IGF1in Proliferation And Differentiation Of Sheep Myoblast

Insulin-like growth factor1(IGF1) is named because its structure is similar to insulin.IGF1not only can promote effectively the proliferation and differentiation of skeletal muscle，enhance the contraction force of regenerating muscle，but also activate the RNA polymeraseactivity，Promote the phosphorylation of non-histone proteins and improve the level ofmRNA，further stimulate the synthesis of RNA and DNA，promote cell growth and finallydrive cells to experience successively each period of the cell cycle. At present，the mechanismthat IGF1how to affect sheep myoblast remain is unclear. In the study，the molecularmechanism that IGF1promotes muscle growth would be discussed by analysing the role ofIGF1in proliferation and differentiation of sheep myoblast.In this study，IGF1gene was cloned and subcloned into the lentiviral expression vectorpLEX-mcs to construct the eukaryotic expression vector，and the cell lines which IGF1stabletransfected sheep myoblast were established，the effect of IGF1on the proliferation anddifferentiation of sheep myoblast when overexpressed IGF1was analysed by the cell growthcurve，flow cytometry and immunofluorescence. Meanwhile，the expression changes of thegene related to proliferation and differentiation in overexpressed IGF1sheep myoblast weredetected by RT-PCR、Realtime PCR，the mechanism of IGF1effect on sheep myoblastproliferation and differentiation was discussed in cell level. This study has obtained data andresults are as follows:1.We cloned the total of518base pair of fragments of full-length coding region of IGF1and overexpressed IGF1in sheep myoblast. The result of Western blotting showed that IGF1was expressed in sheep primary myoblast.2.We isolated and cultured sheep myoblast from fetal muscle tissue by the preplatetechnique. After cell purification and identification，we obtained>90%with high purity sheepprimary myoblast cell lines. 3.The results of cell growth curve indicated that stable expression of IGF1myoblastswere significantly faster than that of the control cell growth (the difference was significant infirst day (P <0.05)，the difference was extremely significant in second to sixth days).Thegrowth curve results of transient transfection of IGF1myoblasts coincided with the stabletransfection；The cell cycle analysis showed the myoblast of stable expression of IGF1earlierinto S phase than control cells，and the cell number in the S phase cells than in control cellsincreased7.77%，and increased the expression of IGF1R and Akt1gene. The results coincidewith the growth curve. These results show that the IGF1can promote the proliferation ofsheep myoblast.4.The results that immunofluorescence assay showed that cell myotube fusion rate andmyotube diameter were respectively30.96%and55.29μm in IGF1overexpression cell line，these datas increased significantly compared to the control cell lines12.26%and25.86μ m，and the fusion rate was increased by60.4%，the diameter also increased nearly doubled，andraised the differentiation gene expression including MyoD and Myogenin. In the presenceof MSTN protein，IGF1transformed cell myotube fusion rate and diameter were10.43%and31.92μ m，while the control group cells myotube fusion rate and diameter were6.09%and23.27μm，the difference was significant (P <0.05). The results show that in the presence ofMSTN， IGF1still can promote the differentiation of sheep myoblast.Above all，this study cloned the total of518base pair of fragments of full-length codingregion of IGF1，lentiviral expression vector pLEX-IGF1was successfully constructed，IGF1stable expression was obtained in sheep myoblast by lentivirus infection, and differentiationand proliferation capacity of the stable infected IGF1cells were evaluated，and also the effectof IGF1gene on differentiation and proliferation sheep myoblast was further verified.