An Experimental Study On Culture Of Rat Satellite Cells In Vivo And Transplantation Of Satellite Cells To Delay Atrophy Of Denervated Muscle

Objective:To explore the techniques of culturing rat satellite cells in vitro, and to observe their proliferation and differentiation in vitro. To observe the survival, differentiation, and formation of myotube of the culured satellite cells in vivo after transplanting them to denervated skeletal muscle, and to judge the effect for delaying atrophy of denervated skeletal muscle by transplantation of satellite cells.Methods:Part Ⅰ:Culture of rat satellite cells in vitro and identification of satellite cells.The hind limb muscles were isolated from adult Wistar rat, disassociated with type I collagenase and trypsin by "two steps method" and purified via differential adhesion. Proliferation medium contained Hams F10+20%FBS+5ng/ml bFGF, differentiation medium contained Ham s F10+2%FBS+2%HS. The proliferation, differentiation and fusion of satellite cells in vitro were observed.Quiescent satellite cells were detected for the expression of Pax7, activated satellite cells were detected for the expression of MyoD, and differentiated satellite cells were detected for the expression of Desmin and α-SCA, by immunofluorescence techniques.Part Ⅱ:Transplantation of satellite cells and observation of their differentiation in vivo.The purity of satellite cells cultured was identified, and then satellite cells were transplanted to denervated gastrocnemius after they were marked with eGFP gene by adenovirus transfection.Gastrocnemius was sampled1week,2weeks and4weeks after transplantation, respectively. And continuous frozen sections of gastrocnemius were produced. The survival, differentiation, and formation of myotubes of the transplanted cells with GFP green fluorescence were observed by fluorescence microscope.Part Ⅲ:Observation of the effect for delaying atrophy of denervated skeletal muscle by transplantation of satellite cells.Animal models for "atrophy of denervated muscle" were established in24adult Wistar rats, by cutting off their right tibial nerves. They were randomly divided into two groups. The right gastrocnemius of one group of12Wistar rats were injected with satellite cells (transplantation group), while the right gastrocnemius of rats in the other group were injected with only culture medium (control group).4weeks after the transplantation, bilateral gastrocnemiuses of six rats from transplantation group and six rats from control group were sampled. After measuring the muscle wet weight, paraffin sections of gastrocnemius were produced for HE staining and Masson staining, then the average cross-sectional area of muscle fiber and hyperplasia of collagen were compared.Right gastrocnemiuses of the other rats were sampled, and frozen sections of gastrocnemius were produced for ATPase histochemistry staining. The content of ATPase of transplantation group and control group were compared.Results:Rat Satellite cells with high purity could be acquired in vitro by digestion with type I collagenase and trypsin, and purification via differential adhesion. Cultured satellite cells could differentiate and mutually fuse to myotubes under right conditions. The quiescent cells expressed Pax7in nucleus, the activated cells expressed MyoD in nucleus, and the differentiated cells expressed Desmin and α-SCA in cytoplasm.Exogenous satellite cells could survive, differentiate and fuse into myotubes in vivo after transplantation.The muscle wet weight, average cross-sectional area of muscle fiber, hyperplasia of collagen fibers and content of ATPase of the transplantation group were all improved, compared with the control group. And the difference was statistically significant.Conclusion:Highly purified satellite cells with potential of muscle regeneration could be acquired in vitro by digestion and separation of rat muscle. The muscle atrophy could be delayed by transplanting the satellite cells in to the denervated skeletal muscle.