| Objective:Proliferation and migration play a crucial role in cellsreaction, the main way through which cell growth factor plays its physiological function is to regulate the proliferation and migration of effector cell. Insulin-like factor I(IGF-I) is an important cytokine which can impact the function of muscle satelite cells. Therefore, it is of practical significance to study the mechanism of IGF-I stimulation on proliferation and migration in muscle satellite cells. mT0R/STAT3 signal pathway is a central controller of cell metabolism and growth, which plays an important role in cellular biotic activities. Also, our early experiments found that STAT3 protein play an important role in IGF-I-stimulated myoblast proliferation. Accordingly, the following experiments attempt to discuss the role of mTOR/STAT3 pathway in IGF-I-stimulated myoblast proliferation and migration.Methods:Part Ⅰ:MTT method was used to examine the OD value of L6 myoblast at 24h, 48h and 72h, which are incubated with 2%FBS, 10ng/mL,50ng/mL, 100ng/mL or 200ng/mL IGF-I respectively. After selecting the proper IGF-I concentration according to the MTT results, RTCA was used to monitor the effect of IGF-I on proliferation of L6 myoblasts. Then the number of transmembrane cell was detected in above groups in 12h to investigate whether IGF-I could promote the migration of myoblast and conclude the optimum concentration for cell proliferation and migration.Part Ⅱ:Refer to the result of Part I, we chose 100ng/mL IGF-I to incubate L6 myoblast (2%FBS as control) and total cellular protein was collected after min, 20min,30min, 1h,2h,4h,12h,24h and 48h after incubation. Then analyzed mTOR/STAT3 pathway activation levels using Western blot assay to find out whether this signaling pathway could be activated after stimulation.Part Ⅲ:Divided cells into three groups:2% FBS group (control),50μmol/L RAPA + 100ng/mL IGF-I group and 100ng/mL IGF-I group. Western blot was used to detect the varies phosphorylation levels of target proteins in each group; MTT assay was used to test changes of myoblast proliferation after 24h,48h and 72h stimulation and RTCA was used to monitor changes of migration ability of myoblast at 12h. Thus we could determine the role of mTOR/STAT3 pathway in IGF-I-stimulated myoblast proliferation and migration.Results:Part Ⅰ:At the same time point, IGF-I of high concentration (50ng/mL, 100ng/mL,200ng/mL) could stimulate L6 myoblast proliferation (p<0.05), among which 100ng/mL and 200 ng/mL IGF-I had a more dramatic effect, while 10ng/mL IGF-I had no significant function (p>0.05). 100ng/mL and 200ng/mL IGF-I could significantly increase L6 myoblast migration (p<0.01), while IGF-I of low concentration (Ong/mL, 10ng/mL and 50ng/mL) has little impact on this.Part Ⅱ:There was no significant differences in total mTOR, STAT3 protein expression between the cells in the experimental group at each time point, while the phosphorylation protein levels (both p-mTOR and p-STAT3) increased along with the the prolongation of action time of IGF-I, which peaked at 4h and then decreased afterwards. However, the phosphorylation level at 48h was still higher than the initial value, which showed statistical significance. Conclusively, mTOR/STAT3 pathway was activated under IGF-I.Part Ⅲ:RAPA, an inhibitor of mTOR/STAT3 signaling pathway, significantly inhibited the IGF-I-stimulated phosphorylation levels of mTOR and STAT3 protein. With the addition of RAPA, the IGF-stimulated L6 myoblast migration was dramatically suppressed, suggesting that IGF-I stimulated proliferation and migration of L6 myoblast through mTOR/STAT3 pathway.Conclusion:IGF-I can promote the proliferation and migration of myoblasts to some extent, this promoting role is related to the activation of the mTOR/STAT3 signaling pathway, but is not the only pathway of activation. |
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