The Effect On Atrophy Process Of Denervated Skeletal Muscle By Transplantation Of Skeletal Muscle Satellite Cell

Research purpose:To explore if the muscle satellite cells can delay the skeleton muscular atrophy.Research Method:1,To prepare muscle satellite cells: Most part of triceps of Wistar rat's forelimb were cut under sterile condition. Two steps enzyme digestion was adapted to separate muscle satellite cells. After discrete gradient centrifugation, purified muscle satellite cells were cultured in vitro. The cells were subcultured for 14-20 passages. When the concentration reached 1×107/ml, they were transplanted. During the cell culturing, differentiation and immunohistochemistry identification were proformed.2,To set up the model of denervated skeleton muscle:16 Wistar rats whose body weight range between 250g to 300g were selected. The tibial nerve of the right leg of each rat was cut with 1cm lesion. And thus the model was made.3,To divide animals into groups:The rats were randomly assigned into two groups, the experimental group and the control group. One day before the transplantation, 0.02% DAPI was added to the cell culture and was incubated overnight as fluorescence label. 0.2ml of muscle satellite cells suspension or physiological saline was injected into the denervated gastrocnemius of the experimental group and the control group respectively.4,To measure and draw material after transplantation :Four weeks after transplantation, under narcosis condition, both groups were stimulated with different strength of electric impulses to identify threshold and optimal value. Then, gastrocnemius from both side were cut off and weighed. Thegastrocnemius was cut in cross direction into to halves. One half was soaked into formaldehyde solution, after 48h, paraffin slides were made in transverse section orientation. HE staining was preformed. The other half was made into frozen slides and observed under fluorescent microscope. Motic Advance 3.2 graphic analytical system was used to measure the cross section area of the muscle fiber.By comparing the damaged and healthy parts of each individual, both experimental group and control group were measured for the recovery rate of threshold of electric impulse stimulation ,the remnant rate of denervated muscle wet weight and the remnant rate of cross section area. The data were shown with average value and standard deviation, SPSS 11.5 statistic software was used for t test for both group.Research Result:Observed under inverted phasecontrast microscope, the newly detached purified muscle satellite cells are round shaped and some refracted. After attach to the dish, they show spindle shape. The cells tend to aggregate together by protruding out to the nearby cells. After they connected together, myotube could be observed.By Immunohistochemistry staining, positive result of desmin expression was observed in mature myotube.In the experimental group, gastrocnemius frozen slides showed clump of fluorescent muscle cell nuclear and muscle fibre, while they were not observed in the control group.Four weeks after transplantation, in experimental group, the recovery rate of threshold of electrical impulse stimulation,the remnant rate of denervated muscle wet weight and the remnant rate of cross section area were compared with that of control group. The differences were statistically significant (P<0.01).Conclusion:In this research, it was demonstrated that transplantation of muscle satellite cells into denervated muscle can facilitate neogenesis of skeleton muscle, maintain the muscle weight, cross section area and muscle tension, and thus alleviate the rate of atrophy of skeleton muscle, and help the recovery of the muscle's function. This may provide a clinical effective way to prevent and treat denervated skeleton muscle atrophy.