Effects Of Mechano Growth Factor On The Proliferation Of Rat Skeletal Muscle Satellite Cells And Akt Expression

PurposeSkeletal muscle satellite cells are kind of muscle-derived stem cells with potential ability of differentiation and proliferation. Activated satellite cells can keep continued proliferation, differentiation, and ultimately fusing in the damage muscle or generating new muscle fibers. IGF-1 has been confirmed playing an important role in the regulation of skeletal muscle hypertrophy. More and more researches found that MGF, one of the isomers of IGF-1, increased significantly after exercise and muscle injury. And it can promote the cultured skeletal muscle satellite cell proliferation and differentiation in vitro. However, we still don't know the proper concentration of MGF on skeletal muscle satellite cell proliferation in vitro. And on the research of the mechanism of proliferation of satellite, more focus now is paid on the Akt signal transduction pathway. In the study, we cultured the rat skeletal muscle satellite cells in vitro and supplied them with five different concentrationes of MGF to find the proper concentration of MGF. Then we used the method of Western Blot to investigate the expression of Akt in all five groups.MethodsWe isolated the leg and the back muscles of a 4-week-old male SD rat in sterile conditions. Satellite cells were obtained by two-step digestion method and velocity sedimentation method, seed in cell culture bottle. Then the anti-desmin was used to identify the satellite cells. Adjust the density; we were divided the satellite cells into five groups according to the concentration of MGF:Ong/ml,25ng/ml, 50ng/ml, 100ng/ml and 150ng/ml. The cells were incubated 96h with different concentration of MGF. A method of CCK-8 was used to test the proliferation of the five groups every 24 hours. The total protein content of the satellite cells was test by the method of BCA. Akt and p-Akt was tested by the method of Western Blotting.Results1. Muscle satellite cells primary culture:after isolated, satellite cells scattered in the bottom in the shape of round and have a strong cell refractive index. 24 hours later, the majority of cells were adhered and the adhesion completed in 48 hours. By then, the adherent cells were irregular in shape, and protruding could be seen in the microscope. The cells connection can be seen in the 96 hours. And the cells are in the shape of spindle. The cell density increased, too. In 144 (6d) hours, some cells differentiated into myotubes. Anti-desmin showed that the purity of muscle satellite cells was 95%. And the cells could be used for following research.2. Satellite cell proliferation test:compared with the control group,25ng/ml and 50ng/ml groups promoted skeletal muscle satellite cell proliferation within a certain time, especially the 25ng/ml group could promote cell proliferation within 24h to 96h (P<0.01) significantly. While the 50ng/ml group enhanced cell proliferation within 24h to 72h (P<0.01). However, both the 100ng/ml and 150ng/ml groups had no change compared with the control group.Viewed from the different time points:compared with Oh, only the 25ng/ml group showed significant increase in the first 24h stage (P<0.01). While between the 48h and 72h stage, both 25ng/ml and 50ng/ml groups had a great increase (P <0.01), particularly in the 72h stage, the two groups reached the highest OD value. In the 96h stage, only the 25ng/ml group increased (P<0.05), but had a less OD than the 72h stage.The BCA test showed that 25ng/ml group had a significantly higher total protein content than the control group (P<0.01). While the 50ng/ml group also increased compared with the control group (P<0.05). And the results were consistent with Western Blot.The Akt and p-Akt results had a significant raise in 25ng/ml (P<0.01) and 50ng/ml (P<0.05) group compared with the control group.Conclusions1. Two-step digestion and velocity sedimentation method was used to isolate skeletal muscle satellite cells. Anti-desmin test showed that the methods were successful for the skeletal muscle satellite cells release.2. In the study, concentrations of 25ng/ml and 50ng/ml of MGF on skeletal muscle satellite cells had a great promotional effect on the proliferation. And MGF-induced muscle satellite cell proliferation might be through the Akt signaling pathway.