Objective To analyze the expression changes of Pax7 and MyoD in satell ite cells of denervated skeletal muscle at the mRNA and protein level, and to explore the pathway concerning with denervation and regeneration of skeletal muscle. Methods Nine male SD rats of 2 months old, weighing 180-200 g were selected. The right gastrocnemius musclewas made denervation model as experimental sides, and the same operation was performed in the left sides as control. The immunofluorescence analysis was done for detecting the protein expressions of Pax7 and MyoD in muscles denervated 3, 7 and 21 days after operation. The RT-PCR analysis was used to explore the mRNA expression of MyoD and Pax7 in extracted tissues from denervated muscles. Results Nine rats survived after operation, with l imited motion of right hind l imbs, and normal motion of left l imbs. The result of immunofluorescence with the polyclonal antibody against MyoD and the monoclonal antibody against Pax7 showed that the number of MyoD positive cells in prolonged denervated muscles (experimental sides) increased gradually, and reached the peak 21 days after denervation, there was significant difference compared with that of control sides (P lt; 0.05). However, the total number of Pax7 positive cells decreased with the prolongation of denervation, the expression of Pax7 was b only at 3 days, then decreased gradually, and there was no positive cells in denervated muscles of 21 days. There were significant differences in Pax7 positive cells between experimental sides and control sides at 3 and 7 days (P lt; 0.05). The results of immunostaining showed that MyoD mRNA levels increased , and reached the peak at 21 days of denervation, there was significant difference compared with that of control sides (P lt; 0.05). Whereas, Pax7 mRNA expressed only in control, denervated muscles of 3 and 7 days, and its expression level decreased gradually. Conclusion One possible reason of the depletion of satell ite cell pool in prolonged denervated muscle is the down-regulation of Pax7.
OBJECTIVE: To construct eukaryotic expression vector of rat myogenin gene for further study on its functions in skeletal muscle denervated atrophy and repair. METHODS: The cloning vectors (containing full length of myogenin cDNA and two restriction sites: Hind III and Xho I) were first cut by two restriction endonuclease: Hind III and Xho I, and the same as the eukaryotic expression vector; then, the myogenin cDNA and the digested vector were ligated by T4 DNA ligase, and recombinant eukaryotic expression vector was formed. Its length was certificated by agarose gel electrophoresis analysis, digestion with Hind III and Xho I, PCR; and the rightness of the myogenin cDNA sequence was confirmed by sequencing. RESULTS: The results of agarose gel electrophoresis analysis, digestion, and PCR confirmed the right length of inserted DNA, which was the same as the myogenin cDNA, and the sequencing result of pcDNA3-myogenin was identical with the reported. CONCLUSION: pcDNA3-myogenin a eukaryotic expression vector, is successfully constructed.