【作者】 徐磊；

【导师】 张凯伦；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 第一部分大鼠骨骼肌成肌细胞体外培养的实验研究目的改进大鼠原代骨骼肌成肌细胞的体外培养方法,得到更高纯度的成肌细胞。方法采用成年Louis近交系大鼠,将两步消化法加以改进,以Ficoll分离和差速贴壁法两步纯化得到更高纯度的骨骼肌成肌细胞,所得细胞采用免疫组织化学方法加以鉴定。结果此方法培养的骨骼肌成肌细胞纯化率可达到98%。细胞增殖快,生长良好。结论本实验成功建立了大鼠原代骨骼肌成肌细胞培养纯化方法,适用于心外科以及相关科室开展细胞移植和基因治疗方面的研究。第二部分血管内皮生长因子基因体外转染大鼠骨骼肌成肌细胞的实验研究目的研究血管内皮生长因子基因(VEGF)在原代培养大鼠骨骼肌成肌细胞中的转染表达,为进一步利用经基因转染的骨骼肌成肌细胞移植治疗心肌梗死的研究打下基础。方法利用分子生物学技术构建VEGF重组真核表达载体,通过脂质体介导将VEGF基因转染到原代培养的大鼠骨骼肌成肌细胞中;经RT-PCR、Western-Blot及免疫荧光检测转基因成肌细胞的基因表达情况。结果成功构建VEGF重组真核表达载体,骨骼肌成肌细胞在基因转染后24h即出现VEGF的表达,于48～72h达表达高峰,以后逐渐下调,维持表达2周左右;转染后的骨骼肌成肌细胞表达VEGF蛋白质产物明显增加。结论经脂质体介导转染VEGF基因的大鼠骨骼肌成肌细胞在体外有良好的基因和蛋白表达,为VEGF基因修饰骨骼肌成肌细胞移植治疗心肌梗死奠定了基础。第三部分低氧环境下HRE对大鼠骨骼肌成肌细胞转染表达VEGF基因的调控作用的实验研究目的研究低氧环境下低氧反应元件(HRE)作为低氧特异性启动子对血管内皮生长因子基因(VEGF)在原代培养大鼠骨骼肌成肌细胞中转染表达的调控作用,为进一步利用经基因转染的骨骼肌成肌细胞移植治疗心肌梗死的研究打下基础。方法利用分子生物学技术,以HRE作为低氧特异性启动子,构建pEGFP- C3- 9HRE– CMV -VEGF重组真核表达载体,通过脂质体介导将其转染到原代培养的大鼠骨骼肌成肌细胞中,在不同低氧浓度及不同缺氧时间下培养,通过RT-PCR、ELISA及荧光显微镜检测不同条件下转基因成肌细胞VEGF基因的表达情况,以判定HRE低氧启动子的功能。结果低氧浓度组可见明显目的基因条带,且随着氧浓度的降低及缺氧时间的延长,目的基因表达增强;低氧环境下,转染后的大鼠骨骼肌成肌细胞表达VEGF蛋白产物明显增加,且随着氧浓度的降低及缺氧时间的延长,VEGF蛋白产物表达亦增强;转基因成肌细胞低氧环境下可见报告基因EGFP表达,常氧环境下未见报告基因表达。结论以多拷贝HRE构建低氧启动子插入VEGF基因上游,可作为控制VEGF基因表达的开关,这对于防止VEGF基因转染的成肌细胞移植后VEGF基因过度表达所引起的安全问题,提高基因治疗的安全性有重要意义。第四部分HRE操控VEGF表达协同骨骼肌成肌细胞移植治疗急性心肌梗死的研究目的研究转染重组真核表达载体pcDNA3.1/VEGF及pEGFP- C3-9HRE–CMV - VEGF的骨骼肌成肌细胞移植对急性心肌梗死的治疗作用,为其临床应用打下基础。方法结扎大鼠冠状动脉前降支建立急性心肌梗死模型,分别将无血清培养基、未转染骨骼肌成肌细胞、转染pcDNA3.1/VEGF的成肌细胞以及转染pEGFP- C3- 9HRE-CMV-VEGF的成肌细胞注射到梗死区,4周后检测死亡率、心脏功能、心肌梗死面积、移植细胞形态、梗死区VEGF表达及毛细血管密度的变化,以阐明转基因骨骼肌成肌细胞对急性心肌梗死的治疗作用。结果转基因骨骼肌成肌细胞移植后,在上述指标方面均优于对照组及未转染成肌细胞移植组:死亡率及心肌梗死面积明显下降,心功能明显改善,梗死区VEGF表达及毛细血管密度明显增加(P<0.01);两个转染组之间无明显差异(P>0.05)。结论转基因骨骼肌成肌细胞移植后对急性心肌梗死的治疗作用明显优于对照组及未转染成肌细胞,移植后转基因成肌细胞分泌VEGF明显增强,可以促进血管发生与形成,从而有效的建立一种有助于移植细胞增殖、分化和形成功能的微环境,证明了转基因骨骼肌成肌细胞移植治疗心肌梗死的可行性。更多还原

【Abstract】 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.更多还原