Research On The Role Of Mechano Growth Factor On The Proliferation Of Skeletal Muscle Satellite Cells And The Signaling Pathway

Objective:Studies have shown that satellite cells and some growth factors play an important role in the muscle growth, adaptation, repairment and transplantation. But the number of activated skeletal satellite cells is inadequate, what's more, the skeletal muscle satellite cells tend to merge. So the key is to make skeletal muscle satellite cells reach sufficient number. As a subtype of IGF-1, mechano growth factor (MGF) can promote proliferation and inhibit differentiation. Through researching the role of MGF on the proliferation of skeletal muscle satellite cells and the signaling pathway, the utilization of exogenous MGF maybe provide a new way to cure skeletal muscle disease in future.Methods:One healthy male SD rat was supplied by Experimental Animal Center, second military medical university. In order to get a higher purity of skeletal muscle satellite cells, this study adopted an improved two-step enzymatic digestion method combined with differential attachment technique. At the third passage, skeletal satellite cells were treated with MGF at final concentration of 15,25,50,100, 200ng/ml. Cells received 100μl growth medium as control group, and those treated with 100μl DMEM cells as negative controls. CCK-8 was added at 24h of culture. CCK-8 assay was utilized to detect the most appropriate concentration promoting cell proliferation of MGF. BCA assay was utilized to detect the total protein, and PI assay was utilized to detect the apoptosis. And immunocytochemical method was used to identify skeletal muscle satellite cells. Then, took the 3rd generation of SCsto divide into growth medium, DMEM control group and experimental group (MGF, LY+ MGF, PD+MGF, LY+PD+MGF). Every group consisted of 6 samples. All SCs were cultured in serum-free growth medium for 1 day, and the corresponding groups added the PI3K inhibitor LY294002 (30μM) and the MEK inhibitor PD98059 (50μM) into the growth medium individually. Changed into growth medium with MGF (25ng/ml) to continue to culture after 12h, and checked the CK-MM concentrations by ELISA to observe the differentiation of SCs after 24h.Results:(1) Compared with the control group, other concentrations of MGF except 200ng/ml had significant proliferation effects on SC(p<0.01). At 25ng/ml, the proliferation promotion effect nearly reached a peak.(2) Since the sixth day, the speeds of cell proliferation in the control group had slowed down evidently. Then, SCs were stepped into the proliferative phase of the platform. Compared with control group, the SC growth curve of experimental group left, the doubling time and the flat-top period shorten.(3) Compared with contradistinctive group, MGF (25ng/ml) had a more significant multiplier effect on skeletal muscle satellite cells at 48 hours (p<0.05),especially at 72 hours (p<0.01). Compared with the contradistinctive group, MGF (25ng/ml) had a more evident effect on increasing their total protein at 48h and 72h (p<0.05). Compared with contradistinctive group, we could conclude that MGF (25ng/ml) had an evidently negative effect on apoptosis of skeletal muscle satellite cells at 48h (p<0.05) and at 72h (p<0.01).(4) Compared with MGF group, the CK-MM expression (p<0.05) of cells of LY+ PD+MGF group had been inhibited markedly, however, the expression of cells of LY+MGF group and PD+MGF group had not changed evidently. Compared with the MGF group, the CCK-8 OD values (p<0.01) of PD+MGF group and LY+PD+MGF group had dropped significantly, however, LY+MGF group had not changed evidently.Conclusions:(1) MGF can promote proliferation of rat skeletal muscle satellite cells in vitro in a dose dependent fashion.25ng/ml is a good concentration for SC culture.(2) The concentration of MGF (25ng/ml) might promote skeletal muscle satellite cells to entry into the plateau earlier and shorten the growth cycle.(3) The concentration of MGF (25ng/ml) might inhibit apoptosis of skeletal muscle satellite cells.(4) The signaling pathway of MEK/Erk might play a more important role on the proliferation of SCs under the utilition of MGF. However, the joint use of PD98059 and LY294002 might inhibit the proliferation of SCs more apparently, because of the intracellular signal cross-cutting.