An Experimental Study On Preventing Denervated Skeletal Muscle Atrophy By Myogenin Gene Transfer

Objective: (1) To clone full-length rat myogenin cDNA, to constructrecombinant cloning vector and to construct eukaryotic expression vector for furtherin vivo gene transfection. (2) To detect whether the eukaryotic expression vector of ratmyogenin gene can properly express moygenin protein in the transient transfectionexperiment, and to investigate whether stable myogenin gene transfection cantransform the primary cultured fibroblasts derived from denervated skeletal muscleinto myoblasts. (3) To assess the value of myogenin gene transfer in the prevention ofdenervated skeletal muscle atrophy by direct injecting eukaryotic expression vectorinto denervated skeletal muscle, and to provide a new way of preventing denervatedskeletal muscle atrophy. Methods: Full-length myogenin cDNA were amplified by RT-PCR, and the PCRproducts were ligated into Hind III/Xho I digested vector pGEM-T to generate therecombinant plasmid, designated pGEMT-moygenin, which was then by sequencingverified. The pcDNA3-myogenin expression plasmid was generated by subcloning themyogenin cDNA into Hind III/Xho I digested vector pcDNA3, the recombinantpcDNA3-myogenin were then transient transferred to primary culture fibroblastderived from denervated rat skeletal muscle by SuperFect transfection reagent.Expression of myogenin was detected by Western blot. Stable transfected cellpopulation was generated by culturing the cells in the presence of G418(800μg/ml)for 3 weeks. Immunofluorescence was used to detect whether stable transfected cellswere transformed into myoblast. That whether transformed cell can proliferate,differentiate and form myotube was also investigated under microscope. After ratsciatic nerve was cut off for 4 weeks, pcDNA3-myogenin(200μg) was directlyinjected into denervated triceps leg muscle and sciatic nerve was sutured. Rats wereeuthanized 8 weeks after transfection. Muscle wet weight, muscle fibercross-sectional area, muscle single contraction strength and muscle tetanuscontraction strength were determined and compared between normal rats, denervatedrats that transfered with pcDNA3-myogenin, denervated rats transfected withpcDNA3 vector alone and denervated rate treated with saline. The differences 4复旦大学博士学位论文(英文摘要)between groups were analyzed by ANOVAF with Tukey test as post hoc test.Myogenin mRNA level and protein level were detected by RT-PCR and Western blotrespectively at 0, 2, 4and 8 weeks after pcDNA3-myogenin transfer in denerved ratstransfected with pcDNA3-myogenin and deverved rats transfected with pcDNA3vector alone. The differences between groups were analyzed by Student T test. Result: PCR, Hind III/Xho I digestion and agarose gel electrophoresis confirmedthe right length of inserted cDNA, and the sequencing results of pGEMT-myogeninand pcDNA3-myogenin were identical with reported rat myogenin cDNA sequence.The immunohistochemistry staining of Vimentin and Desmin confirmed that theprimary cultured cells were fibroblasts and there was no myoblast contamination.Myogenin expression was detected by Western blot 0, 24, 36, 48, 60 and 72 hoursafter transient transfection. The expression was peaked at 60 hours. Stable tranfectedcells were selected in the presence of G418, monoclonal cells were generated 3 weeksafter selection, and immunofluorescence of Desmin confirmed these monoclonal cellswere myoblasts. These transformed cells proliferated normally in the growth mediumand differentiated into myotube 24 hours after applying the differentiation medium.Matured myotubes were observed 5 days after differentiation. During the experiment,myogenin mRNA level and protein level in the skeletal muscle transfected withpcDNA3-myogenin were relatively high in 4, 6, 8, 12 weeks, while myogenin mRNAlevel and protein level was decreased in 6, 8, 12 weeks in the skeletal muscletransfected with pcDNA3 vector alone. Muscle wet weigh...