Experimental Study On The Therapy Of Acute Myocardial Infarction With The Transplantation Of Skeletal Myoblasts Transfected With Vascular Endothelial Growth Factor Gene Regulated By Hypoxic Response Elements

PartⅠCulture of Primary Rat Skeletal Myoblasts in VitroObjective To improve the culture method of primary rat skeletal myoblasts in vitro and obtain more purified skeletal myoblasts.Methods The more purified skeletal myoblasts from Louis inbred strain rats isolated by improved two-step digestion method, and purified by the two-step purification method of Ficoll separation and velocity sedimentation. The cells were identified with immunohistochemistry stain.Results The purification rate of skeletal myoblasts was 98% with the improved method and the cells showed satisfactory growing state and strong proliferative activity.Conclusion The research improved the purification technique for skeletal myoblasts successfully. It was useful for cardiac surgery department or other relative departments to study cell transplantation and gene therapy. PartⅡExperimental Study on the Transfection and Expression ofVascular Endothelial Growth Factor Gene inPrimary Cultured Rat Skeletal MyoblastsObjective To investigate the transfection and expression of vascular endothelial growth factor gene in primary cultured rat skeletal myoblasts.Methods VEGF165 gene was reconstructed in pcDNA3.1 vector and transfected into primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western-Blot and immunofluorescent staining.Results pcDNA3.1-VEGF vector was reconstructed successfully. Transfected myoblasts expressed VEGF165 gene in 24 hours, and the peak appeared in 48～72 hours, and the transfected myoblasts could continuously express gene during 2 weeks. Transfected myoblasts could express protein products in a higher level.Conclusion Skeletal myoblasts transfected with VEGF gene by lipofectamine could express gene products efficiently in vitro, which may contribute to the transplantation of skeletal myoblasts transfected with VEGF gene for the therapy of myocardial infarction. PartⅢExperimental Study on the Regulation of Hypoxic Response Elements to the Expression of Vascular Endothelial Growth FactorGene transfected to Primary Cultured Rat SkeletalMyoblasts in Hypoxic EnvironmentObjective To investigate the regulation of hypoxic response elements to the expression of vascular endothelial growth factor gene transfected to primary cultured rat skeletal myoblasts in hypoxic environment.Methods pEGFP-C3-9HRE-CMV-VEGF vector with HRE as specific hypoxia promoter was constructed by molecular biology technique and transfected into primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, ELISA and fluorescence microscope with different oxygen concentration and different hypoxia time.Results Transfected myoblasts expressed VEGF gene and protein products in hypoxic environment and in a higher level with lower oxygen concentration and longer hypoxia time. EGFP expressed only in hypoxic environment and the expression is unavailable in normoxic environment.Conclusion Specific hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene in hypoxic and normoxic environment, which could enhance the reliability of gene therapy. PartⅣExperimental Study on the Therapy of Acute Myocardial Infarction with the Transplantation of Skeletal Myoblasts Transfected with Vascular Endothelial Growth Factor Gene Regulatedby Hypoxic Response ElementsObjective To investigate the therapy of acute myocardial infarction with the transplantation of skeletal myoblasts transfected with pcDNA3.1/VEGF vector and pEGFP-C3-9HRE-CMV-VEGF vector.Methods The rat models of acute myocardial infarction were established with the occlusion of anterior descending coronary artery. Serum-free medium, control skeletal myoblasts, skeletal myoblasts transfected with pcDNA3.1/VEGF vector and skeletal myoblasts transfected with pEGFP-C3-9HRE-CMV-VEGF vector were injected into the border zone surrounding the infarct (4 injections of 1x106 cells in 100μl) respectively. The mortality, cardiac function, infarct size, grafted myoblasts, expression of VEGF and angiogenesis were observed 4 weeks later to demonstrate the therapy of acute myocardial infarction with transfected skeletal myoblasts.Results These indicators were better in the two groups of transfected skeletal myoblasts than in the groups of medium and control skeletal myoblasts. The mortality and infarct size were significantly reduced, and the cardiac function, the expression of VEGF and angiogenesis were increased improved(P<0.01).Conclusion This combined strategy of skeletal myoblasts transplantation with gene therapy could be of importance for the treatment of acute myocardial infarction.