| OBJECTIVEDuring myogenesis, skeletal muscle satellite cells play a key role in the process of repair and renewal of myofibers. Studies have shown that MGF appears to initiate muscle satellite cell activation and proliferation in vivo. And based on that, it has been observed the effects of MGF on activation and the dose-dependent manner on proliferation of SC in vitro which increasing the number available for local repair, as well as the mechanism at the levels of molecular.METHODSThe primary passage SC were derived from gastrocnemius muscle and soleus muscle of the Sprague-Dawley rats (4 weeks of age, male).-The SC were digested with 0.1% CollagenaseⅡand 0.25% Trypsin, purified with means of differential attachment technique and identified withα-sarcometric actin.The third passage SC were treated with MGF at five different doses:lOng/ml, 25ng/ml,50ng/ml, 100ng/ml,200ng/ml. The cell proliferation rate was measured by MTT cell proliferation assay after 24h,48h,72h,96h and the best dose of proliferation was determined. The third passage SC were serum starved for 24h without FBS and then were divided equally into experimental groups for different time points and one operated group treated with MGF plus one normal control group cultured with DMEM. The cell cycle was detected with Flow Cytometry after 8h, 16h,24h and 32h.RESULTS1. Assessment of cell viability showed that the live SC proportion was 98.8%.2. The immunocytochemistry showed that the proportion of a-sarcometric actin positive SC was 97.4%.3. The proliferation of SC was positively correlated with the MTT A value. 48h:The A values of the operated groups which treated with MGF of 25ng/ml and 50ng/ml were significantly higher than that of normal control group (P<0.01). The A value of 10ng/ml group was higher than that of normal control group (P<0.05). There was no significant difference between 100ng/ml group and normal control group (P>0.05) and the same effect was seen between 200ng/ml group and normal control group (P>0.05). There was no significant difference between 100ng/ml group and 200ng/ml group (P>0.05). 96h:The A values of operated groups of 25ng/ml.50ng/ml. 100ng/ml and 200ng/ml were significantly higher than that of 10ng/ml group as well as normal control group (P<0.01). There was no significant difference among the operated groups of 25ng/ml.50ng/ml, 100ng/ml and 200ng/ml (P>0.05).There was no significant difference between 10ng/ml group and normal control group (P>0.05). With the dose increasing, the proliferation peaked ranging from 25ng/ml to 50ng/ml and was inhibited thereafter. As time went by, the proliferation was inhibited at 96h with the treatment of MGF. Combining the time with the dose, it could be observed that there was an interaction effect between 25ng/ml group and 50ng/ml group at 48h.4. The proportion of SC in GO phase was 86.76% by serum starvation with serum-free DMEM for 24 hours.5. After 8h and 16h incubation there was no significant difference between the operated group and the normal control group (P>0.05). And there were significant differences between the operated group and the normal control group, the proportion of the SC in G0/G1 phase of operated group treated with 25ng/ml MGF was significantly lower than that in normal control group after 24h (P<0.01) as well as 32h (P<0.05).CONCLUSIONS1. MGF increases skeletal muscle SC proliferation.2. MGF promotes skeletal muscle SC to proliferate in a dose-dependent and a time-dependent manner. And the ideal range of dose may be from 25ng/ml to 50ng/ml. SC begin to proliferate at 24h with the treatment of MGF.Serum starvation without FBS can synchronize SC in GO phase within 24h.3. MGF appears to initiate skeletal muscle SC activation of 4-week-old Sprague-Dawley rats. |
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