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为探讨人载脂蛋白AI和卵磷脂胆固醇酰基转移酶基因在肌源性细胞中异源共表达的可能性 ,构建含上述基因和新霉素磷酸转移酶基因的多顺反子重组逆转录病毒载体 ,以此制备重组病毒颗粒并转染小鼠原代肌母细胞及C2C12肌源性细胞株。酶联免疫吸附法和免疫组织化学检测证实转染后的细胞均具有异源共表达人载脂蛋白AI与卵磷脂胆固醇酰基转移酶的能力。经G418筛选则获得稳定转化的C2C12细胞株 ,6 0天后仍能有效共表达人载脂蛋白AI与卵磷脂胆固醇酰基转移酶。聚合酶链反应法检测显示人载脂蛋白AIcDNA与IRES序列均有效整合于靶细胞基因组中。提示以重组逆转录病毒为载体在体外对肌源性细胞进行遗传修饰 ,再移植回骨骼肌使之在体内长期高效表达载脂蛋白AI和卵磷脂胆固醇酰基转移酶 ,可能是一种值得探讨的通过促进胆固醇逆转运途径来防止或减轻高脂血症和动脉粥样硬化的方法。
In order to investigate the possibility of heterologous co-expression of human apolipoprotein AI and lecithin cholesterol acyltransferase gene in myogenic cells, a polycistronic recombinant retrovirus containing the above gene and the neomycin phosphotransferase gene was constructed Vector to prepare recombinant virus particles and transfected mouse primary myoblast and C2C12 myogenic cell lines. Enzyme-linked immunosorbent assay and immunohistochemistry confirmed that all the transfected cells had heterologous expression of human apolipoprotein AI and lecithin cholesterol acyltransferase. Stably transformed C2C12 cell line was obtained after G418 selection, and 60 days later, it was still effective to co-express human apolipoprotein AI and lecithin cholesterol acyltransferase. Polymerase chain reaction assays showed that both human apolipoprotein AIcDNA and IRES sequences were efficiently integrated into the target cell genome. Suggesting that the use of recombinant retroviral vector as a vector to genetically modify myogenic cells in vitro, and then transplanted back to skeletal muscle so that the long-term high-level expression of apolipoprotein AI and lecithin cholesterol acyltransferase in vivo may be a worthwhile study Methods to prevent or reduce hyperlipidemia and atherosclerosis by promoting the cholesterol translocation pathway.