Effects Of Igf-Ⅰ And MGF On The Proliferation And Migration Of Muscle Satellite Cells In Vitro

OBJECTIVEDuring myogenesis, skeletal muscle satellite cells play a key role in the process of repair and renewal of myofibers. In the unperturbed state, skeletal muscle satellite cells remain in a nonproliferative, quiescent state. In response to stimulation, the satellite cells become activated, proliferate, emigrate to the damaged region and fuse to the existing myofiber or fuse together themselves first, then repair the damaged region. In order to discover the relationship between the IGF-I/MGF and skeletal muscle satellite cell regeneration, this research used rat skeletal muscle satellite cell culture, examined the effects of IGF-I and MGF on the proliferation and migration of muscle satellite cells (SC).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% Collagenase II and 0.25% Trypsin, purified with means of differential attachment technique and identified withα-sarcometric actin. The third passage SC were treated with IGF-I and MGF at five different doses: 15ng/ml, 25ng/ml, 35ng/ml, 45ng/ml, 55ng/ml. The cell proliferation rate was measured by CCK-8 cell proliferation assay after 24h, 48h, 72h, 96h and the ideal dose of proliferation was determined. The cell migration rate was measured by Transwell assay after 14h and the ideal does of migration was determined.RESULTS1. Assessment of cell viability showed that the live SC proportion was 97.8%.2. The immunocytochemistry showed that the proportion ofα-sarcometric actin positive SC was 96.9%.3. The proliferation of SC was positively correlated with the CCK-8 OD value. IGF-I: at 24h, 15ng/ml, 25ng/ml, 35ng/ml were significantly higher than normal control group (P<0.05, P<0.01, P<0.01). At 48h, 25ng/ml group and 35ng/ml group were significantly higher than normal control group (P<0.01). At 72h, 15ng/ml group, 25ng/ml group, 35ng/ml group were significantly higher than normal control group (P<0.01, P<0.01, P<0.05). At 96h, 15ng/ml group, 25ng/ml group were high than normal control group and 35ng/ml group (P<0.05). MGF: at 24h, 15ng/ml, 25ng/ml, 35ng/ml and 45ng/ml were significantly higher than normal control group (P<0.01, P<0.01, P<0.01, P<0.05). At 48h, 15ng/ml, 25ng/ml, 35ng/ml were significantly higher than normal control group (P<0.05, P <0.01, P<0.01). At 72h, 15ng/ml, 25ng/ml, 35ng/ml were significantly higher than normal control group (P<0.01). At 96h, there was no significant difference between 15ng/ml, 25ng/ml, 35ng/ml and normal control group (P>0.05).4. The migration of SC was positively correlated with the Transwell OD value. IGF-I: After 14h, there was no significant difference between 15ng/ml, 35ng/ml and normal control group (P>0.05); 25ng/ml, 45ng/ml, 55ng/ml were significantly higher than normal control group (P<0.01), 25ng/ml was significantly higher than 45ng/ml group (P<0.01), there was no significant difference between 55ng/ml and 45ng/ml group (P>0.05).MGF : After 14h, there was no significant difference between 15ng/ml, 35ng/ml and normal control group (P>0.05); 25ng/ml, 45ng/ml, 55ng/ml were significantly higher than normal control group (P<0.01), 45ng/ml and 55ng/ml were significantly higher than 25ng/ml group (P<0.01, P<0.05). CONCLUSIONS1. IGF-I and MGF can promote skeletal muscle satellite cell proliferation. They promote SC to proliferate in a dose-dependent and a time-dependent manner. The ideal range of dose of IGF-I is from 15ng/ml to 35ng/ml, MGF is from 25ng/ml to 35ng / ml. SC begin to proliferate at 24h with the treatment of them.2. IGF-I and MGF can promote skeletal muscle satellite cell migration. The ideal dose of IGF-I is 25ng/ml, MGF is from 45ng/ml to 55ng/ml.