逆转录病毒载体介导人载脂蛋白与卵磷脂胆固醇酰基转移酶基因在骨骼肌的异源性共表达

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,60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AI cDNA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。

Ectopic Co-Expression of Human Apolipoprotein AI and Lecithin Cholesterol Acyltransferase in Mice Skeletal Muscle Cells Introduced by Retroviral Vectors

Aim To investigate the possibility of ectopic expression of apolipoprotein AI (apo AI) and lecithin cholesterol acyltrasferase(LCAT) by myogenic cells and developing a new approach of gene therapy for atherosclerosis (As). Methods Recombinant replication-deficient viral particles were prepared with polycistronic retrovirus vectors containing apoAI cDNA, LCAT cDNA and the neomycine phosphotransferase gene (NEO), Mice primary cultured myoblasts and myogenic cell line C2C12 were transfected by these viruses. The efficiency of transfection and the state of integration were detected by PCR, while the expression of apo AI and LCAT were measured by ELISA and immunohistochemical method. Results All transfected mice myoblasts and C2C12 cells gained the ability of co-expressing human apo AI and LCAT. Stable transfected C2C12 cell line selected by G418 maintained the ability of co-expressing apo AI and LCAT for 60 days. PCR shown the apo AI cDNA and IRES sequence were integrated into genomes of target cells effectively. Conclusions These finds indicated mouse primary myoblasts and C2C12 myoblasts transduced with recombinant retroviral vectors could efficiently express and secrete human apo AI and LCAT. It suggested that the use of polycistronic retrovirus vectors containing human apo AI and LCAT cDNA to genetically modify myoblasts in vitro and then implantation back to skeletal muscle to high efficiently, long-term express apo AI and LCAT in vivo, might be a strategy to prevent or treat hypercholesterolemia and As by promoting the "reverse cholesterol transport" pathway.