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An Experimental Study on Culture of Rat Satellite Cells in Vivo and Transplantation of Satellite Cells to Denervated Muscle
Li Chen, MD, PhD; Lei Xu, MD, PhD; Jianguang Xu, MD, PhD; Huashan Hospital
Huashan Hospital, Fudan University, Shanghai, China
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.
Methods: 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 Ham`s 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.
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 sampled 1 week, 2 weeks and 4 weeks 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.
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 Pax7 in 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.
Conclusion: Highly purified satellite cells with potential of muscle regeneration could be acquired in vitro by digestion and separation of rat muscle. Transplanted satellite cells could survive, differentiate and fuse into myotubes in vivo.
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