The Effects Of IGF-1and TGF-β1on The Differentiation And Fusion Of The Goat SMSCs And The Expressions Of Genes

Skeletal muscle satellite cells(SMSCs) get more and more attentions as myogenic stem cells. They are playing a very importment role in muscle growth, development and damage repair. In this research, the goat SMSCs were obtained from new born Jianyang Daer goat by tissue separation. We observed the cellular morphology, detected the expressions of Pax7, MyoG, Myf5by semi-RT-PCR and tested the expressions of Desmin and a-sarcomeric actin by immunofluorescence assay to identify the cells. In addition, the expressions of MRFs in the periods of proliferation and differentiation were tested by quantitative RT-PCR. Finally, the cells were treated with IGF-I and TGF-β1respectively and jointly to tested the ability of differentiation and fusion. The results were showed as follows.(1) We observed the cells and found that they were round before adherent, and became oval after two hours culturing,24h latter they became spindle-shaped. When they cultured in the differentiate medium, the myotube was found at3d, and the numbers was increased at4d. The Pax7、MyoG、Myf5genes were expressed in different cells periods. Desmin and a-sarcomeric actin were also expressed in cytoplasm and myotube. All these results indicated that we obtained the SMSCs by tissue separation and the cells cultured in vitro could be induced.(2) The expressions of MRFs changed with time. The MyoD and Myf5were expressed in proliferation periods, up to their highest level at2d and3d, then down-regulated in differentiation periods. The MyoG and Myf6were abundantly expressed in differentiate periods whereas the expressions in proliferation periods were relatively weak. We compared the expressions of the four genes and found that the expression of the MyoD was the highest, and then the Myf5, the expressions of MyoG and Myf6were the lowest in proliferation periods. When the cells were induced, The expression of MyoG was the highest, and then the MyoD, the expressions of Myf5and Myf6were the lowest. We conjectured that the MyoD and Myf5were the primary genes during the proliferation periods, and the functions of MyoG and MyoD were major during differentiation periods of goat SMSCs.(3) The IGF-I significantly increased the fusion ability of satellite cells(P<0.01). The expressions of MyoG and Myf6were up-regulated at3d(P<0.01), and down-regulated at the other times. TGF-β1significantly inhibited the expression of MRFs (except Myf5)(P<0.01), and the fusion index also dropped significantly(P<0.01). When the cells were treated jointly by IGF-I and TGF-β1, the expressions of MRFs were similar to TGF-β1treated. All these results showed that the IGF-I could promote the fusion of goat SMSCs significantly, while the effects of TGF-β1on cells was on the contrary.(4) We studied the influence of IGF-I on Smad2and Smad3and found the expression of Smad2was significant increased at3d(P<0.05), and no influence at the other time. the expression of Smad3was highly significant reduced at3d(P<0.01), and no influence at the other time. The results showed that the influence of IGF-I on the expressions of Smad2and Smad3were weak. The TGF-β1was highly significant increased the expression of Smad.2at1d and3d(P<0.01), and reduced at5d. the expression of Smad3was highly significant reduced(P<0.01). When the cells were treated jointly by IGF-I and TGF-β1, the expressions Smad2and Smad3were similar to TGF-β1treated. All these results showed that TGF-β1could influence the expressions of Smad2and Smad3.