The Change Of MiR-206 And MiR-486 And Their Related Regulatory Factors In Satellite Cells Proliferation And Differentiation

Objective Skeletal muscle injury is one of common injuries in sports training and daily life, but the skeletal muscle repair ability is limited, in the professional athletes, muscle damage can lead to muscle inflammation repeatedly, even muscle fibrosis, and loss of muscle contraction, thus affect the sport ability. After muscle damage,the satellite cells which was in a resting state between the membrane and muscle membrane will be activated, and through the proliferation, differentiation, mutual integration into multi-core tube, repair damage or necrosis of muscle fiber, in order to repair the damaged skeletal muscle.However,it is not so clearly studied about the satellite cell activation, proliferation and differentiation. Previous studies mainly used C2C12 into muscle cell line to study the activation of satellite cells, proliferation and differentiation, but the original generation satellite cells are used to study satellite cell proliferation and differentiation of the less, with the original generation satellite cells to simulate the growth of state will be closer to the vivo study. Micro RNA-206(mi R- 206) and Micro RNA-486(mi R-486) is a high specificity and expressed mi RNAs in skeletal muscle, but it is not clear about the expression and role of satellite cells.In the present study, obtained the satellite cells extracted from SD rats, which were divided into normal culture group and differentiation group, compared the expression condition of Pax7 and Myf5 in different time points or group mi R-206 and mi R-486 and its related factors. And it is aimed to investigate the process of satellite cell proliferation and differentiation, mi R-206 and mi R-486 and related regulatory factor expression changes. We hope to provide new research evidence in enriching and improving muscle regeneration. And it is of great significant to provide effective new way of thinking and methods for skeletal muscle injury.Research Methods Choice of 7-day-old SD rats, after ether anesthesia 75% alcohol soaked disinfection,decapitated, take the gastrocnemius and soleus, dissecting microscope quickly stripped fat, fascia and other organizations; fully minced after using type II collagenase, trypsin two-step enzymatic digestion released satellite cells, fetal bovine digestion was terminated; using differential adherence to purify muscle satellite cells.Take 3-5 generations of satellite cells intervention experiments into two groups:normal group(GM group, high serum culture), differentiation group(DM group, low serum culture). Conduct the following tests:1 Using immunocytochemistry to detect the expression of striated muscle actin(α-SCA), the purity of the satellite cells were identified.2 Using the growth conditions on the fifth day after the enzyme-linked immunosorbent assay, and the expression of normal culture induced differentiation of satellite cells in the first five days of MHC, compared to normal cultured satellite cells and induce differentiation.3 Using the PT-PCR testing differentiate Pax7, Myf5 m RNA changes and changes in skeletal muscle-specific mi R-206 and mi R-486 of the both groups during the 1st, 2nd,3rd, 5th-day.4 Using the Western Blot detection to detect the changes of Pax7, Myf5 protein levels.To ensure the reliability of each step of the experiment, the experiment required to take different batches of cells were repeated three times.Results1 Satellite cells striated muscle actin(α-SCA) immunochemical staining, cells were immunoreactive for satellite, satellite cells results show a higher purity.2 Enzyme-linked immunosorbent assay showed that the induction of differentiation group was higher than normal group in the first five days MHC, induction of differentiation group described a relatively high degree of differentiation.3 PCR results1) In normal culture group, Pax7 m RNA expression decreased, the 2nd, 3rd, 5th day was lower than the first day, and there is a significant difference(P <0.05).Induced differentiation group was decreased, but there was no significant difference between the different time points. There was no significant difference between the two different points of time.2) In normal group, the expression of Myf5 m RNA decreased gradually. In differentiation group, there is no significant differences between time points. In normal group, Myf5 m RNA at various time points is higher than differentiation group,but no significant difference statistically.3) The expression of Mi R-206 in both groups were firstly decreased, and then increased slightly. But there was no significant difference among the same group at each time point.4) The expression of Mi R-486 in both groups was substantially similar with the expression of mi R-206. Firstly drop, then rise slightly. But there was no significant difference among the same group at each time point.4 Western Blot test:1) Expression of Pax7 protein in differentiation and normal group showed that normal group Pax7 protein decreased gradually, and one day, compared protein expression Pax7 first five days significantly decreased(P <0.05). Induced culture group, Pax7 protein decreased gradually, and the first day compared to a significant decrease in the expression of Pax7 protein 3, 5 days(P <0.05). No significant differences between the two groups at the same time point.2) Induced differentiation of normal culture group and Myf5 protein group, the results showed that the normal culture group and induce differentiation group were tested Myf5 protein gradually decreased, but there was no significant difference.Conclusion1) The satellite extracted by the two step enzyme digestion and differential centrifugation have the ability to differentiation and proliferation. And When the satellite cells at high confluence, they will differentiate and fuse to myotube.2) In the satellite cell differentiation process, Pax7 and Myf5 expression decreased, Pax7 promoted Myf5 expression.3) The expression of mi R-206 and mi R-486 are different in the different medium.The expression of mi R-486 and mi R-206 in the proliferation medium was the highest in third day. The expression of mi R-486 and mi R-206 in differentiation medium was the highest in first day. It is speculated that the expression of mi R-206 and mi R-486 are increased in the initial when the satellite fusion into myotubes.