| Muscle injury resulting from various traumata, neurogenic and myogenic myopathy, such as muscular dystrophy, has been long affecting people's life, though there are many therapeutic means including medicine, physical therapy, traditional Chinese medicine and herbs, intervention and so forth. However, because of the complicated causes resulting in muscle injury, the unclear mechanism of pathogenesis, and the limit amount of cells, mainly satellite cells, directly involved in the healing of muscle injury, the outcomes of above-mentioned treatment are not satisfying. Thus, how to repair muscle injury has been considered a heat and difficult issue for a long time.In recent years, gene therapy with mesenchymal stem cells (MSCs) has been studied in the treatment of injury of muscle. As early as 1864, German pathologist Conheim, for first time acclaimed that there exist MSCs in the bone marrow when he studied the healing of lesion, which has been proved in all round in the studies in later dozens of years. MSCs, stem cells derived from the mesoderm, are capable of multi-differentiating, mainly existing in connective tissue in all over the body and organ mesenchyma, especially abundant in the bone marrow. The cells, potential to differentiate into osteoblasts, chondrocytes, lipocytes, myoblasts, marrow stromal cells and neurocytes, are easy to be isolated and cultured, and theoretically, keep the differentiation to be mesenchymal system after 40 passages, so, the cells can be regarded as better target cells for extrogenic gene transference andexpression and optimal vector for cellular gene therapy. MyoD, one main member of myogenic regulatory factors Family because its transfection can initiate the process of muscle differentiation, merely make muscle stern cells differentiate into myoblasts. After the transportation of MyoD-transfected cells, the regenerated muscle has morphology totally in common with myofibers derived from satellite cells. Due to the reasons mentioned above, based on the potential of myoblast differentiation, we transfected an eukaryotic expression bicistron plasmid vector pIRES2-EGFP-MyoD labeled with enhance green fluorescent protein (EGFP) into MSCs and induced the cells differentiate into myoblasts. After proliferative culture, the differentiated MSCs and normal MSCs were respectively grafed into the focuses of muscle injury induced by cardiotoxin. Roles of MSCs and MSCs-differentiated myoblasts were investigated in the reparation of muscular damages in order to explore a novel method to accelerate the regeneration and repair of muscle injury and reduce the rate of disability. The main results are as follows.1. Bicistron plasmid vector pIRES2-EGFP-MyoD expressing in eukaryotic cells was constructed in success. After the MyoD cDNA fragment was cut form original plasmid EMSV with EcoR I and purified with electrophoresis, it was ligated with linearized EcoR I -cut vector pIRES2-EGFP in present of a ligase T4 DNA. The obtained pIRES2-EGFP-MyoD was then transferred into an engineered bacterium DH5a to replicate the target plasmid. After the indentification of insert direction of MyoD cDNA fragment with Hind III digestion, those plasmid vectors with positive insertion were harvested. The fragment sequence was confirmed to be consistent with the reported one in Genbank.2. The plasmid pIRES2-EGFP-MyoD was transfected into MScs by 10.3% with aid of lipotransfection method. After G418 screening, the expression of MyoD was detected in the transfected MSCs with RT-PCR andthe amplified product was in same in sequence with that from Genbank. Fluorescence was observed in the transfected cells under a fluorescent and laser cofocal microscopes, indicating the existence of reporter gene. Immunohistochemical methods indicated the expressions of MyoD, myogenin, myosin, myoglobin and desmin. But they are negative in control. Fluorescent intensity was stronger in the cells with MyoD transfection than those without. Compared with the later, the former had more in the phase of G0/G2...
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