重组多顺反子逆转录病毒介导的人载脂蛋白AI 与卵磷脂胆固醇脂酰基转移酶在肌源性细胞的表达

目的 :载脂蛋白ＡＩ(ａｐｏｌｉｐｏｐｒｏｔｅｉｎ ,ａｐｏＡＩ)与卵磷脂胆固醇脂酰基转移酶 (ｌｅｃｉｔｈｉｎ :ｃｈｏｌｅｓｔｅｒｏｌａｃｙｌｔｒａｎｓ ｆｅｒａｓｅ ,ＬＣＡＴ)作为高密度脂蛋白 (ｈｉｇｈｄｅｎｓｉｔｙｌｉｐｏｐｒｏｔｅｉｎ ,ＨＤＬ)的主要组分 ,在胆固醇由周围组织向肝脏的运输、排泄过程即胆固醇逆转运 (ｒｅｖｅｒｓｅｃｈｏｌｅｓｔｅｒｏｌｔｒａｎｓｐｏｒｔ,ＲＣＴ)过程中起关键作用。近年研究进一步证实 ,ＡｐｏＡＩ与ＬＣＡＴ的转基因动物能纠正由于遗传缺陷所致的低ＨＤＬ血症并有效抵抗高脂食物引起…     

Targeted Expression and Secretion of Human Apoprotein AI, Apoprotein E and Lecithin cholesterol Acyltransferase from Myogenic Cells

Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓａｎｄｉｔｓｃｏｎｓｅｑｕｅｎｃｅｓａｃｃｏｕｎｔｆｏｒｍｏｓｔｍｏｒｂｉｄｉｔｙａｎｄｍｏｒｔａｌｉｔｙｉｎｔｈｅｗｏｒｌｄ.Ａｎｕｍｂｅｒｏｆｅｐｉｄｅｍｉｏｌｏｇｉｃａｌｓｔｕｄｉｅｓｈａｖｅｄｅｍｏｎ ｓｔｒａｔｅｄａｎｉｎｖｅｒｓｅｒｅｌａｔｉｏｎｓｈｉｐｂｅｔｗｅｅｎ ｐｌａｓｍａｈｉｇｈ ｄｅｎｓｉｔｙｌｉｐｏｐｒｏｔｅｉｎ (ＨＤＬ)ｃｏｎｃｅｎｔｒａｔｉｏｎａｎｄｔｈｅｉｎｃｉｄｅｎｃｅｏｆａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ[1 ] .Ｉｔｉｓｗｉｄｅｌｙａｃ ｃｅ…     

Targeted Expression and Secretion of Human Apoprotein AI,Apoprotein E and Lecithin-choles-terol Acyltransferase from Myogenic Cell

Objective To investigate the possibility of heterologous expression for apoAI, apoE and LCAT by skeletal muscle cells and secretion into blood and to develop a safe and convenient gene therapy method for atherosclerosis. Methods Viral and nonviral vectors containing apoAI, apoE or LCAT genes were constructed and transfected into myogenic cells in vitro or injected directed into mouse skeletal muscle. The expression efficiencies of these vectors were investigated by ELISA assay for human apoAI and apoE3 and by the proteoliposome method for human LCAT. Genomic DNA was extracted from stable transduced myoblasts and analyzed for the presence of vector sequence by PCR amplifications. Immunocytochemistry assay was also performed to make an intuitionistic detection for the expression of transgene in myoblasts. Results All viral or nonviral vectors constructed in present study expressed the transgenes efficiently in mice skeletal muscles in vivo or cultured myoblasts in vitro. The transgene expression level of cells transfected with AAV-based plasmid vectors were 2-4 times higher then that of cells transfected with conventional plasmid vectors. Additionally, cells transfected with AAV-based bicistronic vector or tricistronic retroviral vector expressed both human apoAI and LCAT simultaneously. The sequences of retroviral or AAV-based plasmid vectors were found to be retained in host cells after transfection when that of conventional plasmid vectors were lost. Furthermore, transduced myoblasts maintained the ability for heterologous expression of human apoAI and LCAT even after differentiation into myotubes. For cells transfected with retroviral vectors, stable transduced clones can be selected by G418 and continued to efficiently express human apoAI and LCAT for 3 months. Conclusion These finds indicated that mice skeletal muscles or cultured myoblasts transduced with viral or non-viral vectors could efficiently express and secret human apoAI, apoE and LCAT. It suggested that the use of nonviral, adenoviral or AAV-based vectors to directly inject into skeletal muscle or the use of polycistronic retroviral to genetically modify myoblasts ex vivo and then implantation back to skeletal muscle to high efficiently and long-term express apoAI, apoE and LCAT in vivo might be a safe and feasible strategy to prevent or reduce the formation of atherosclerotic lesions.     

重组多顺反子逆转转录病毒介导的人载脂蛋白AI与卵磷？…

目的：载脂蛋白ＡＩ（ａｐｏｌｉｐｏｐｒｏｔｅｉｎ，ａｐｏＡＩ）与卵磷脂胆固醇基转移酶（ｌｅｃｉｔｈｉｎ：ｃｈｏｌｅｓｔｅｒｏｌ ａｃｙｌｔｒａｎｓ－ｆｅｒａｓｅ，ＬＣＡＴ）作为高密度脂蛋白（ｈｉｇｈ ｄｅｎｓｉｔｙ ｌｉｐｏｒｏｔｅｉｎ，ＨＤＬ）的主要组分，在胆固醇由周围组织向肝脏的运输、排泄过程即胆固醇逆转运（ｒｅｖｅｒｓｅ ｃｈｏｌｅｓｔｅｒｏｌ ｔｒａｎｓｐｏｒｔ，ＲＣＴ）过程中起关键作用。     

重组逆转录病毒介导的人载脂蛋白AI基因在肌源细胞中的表达

目的 探索一种安全有效的载脂蛋白ＡＩ基因表达系统。方法 构建含人载脂蛋白ＡＩ ｃＤＮＡ的双顺反子逆转录病毒载体ｐＬＡＥＮ,用其转化小鼠原代肌母细胞及肌源性细胞Ｃ２Ｃ１２,ＥＬＩＳＡ法检测细胞液中人载脂蛋白ＡＩ的含量。结果 转化后的小鼠原代肌母细胞及Ｃ２Ｃ１２细胞在分化为肌管细胞后均获得异源表达人载脂蛋白ＡＩ的能力;而且经Ｇ４１８筛选也获得稳定转化的Ｃ２Ｃ１２细胞株,３０天后仍保持有效表达人载脂蛋白     

脂蛋白代谢紊乱的基因治疗研究进展

由血浆脂蛋白的合成、加工或分解代谢异常引起的脂蛋白代谢紊乱大多与遗传因素有关。利用逆转录病毒、腺病毒等载体,通过体细胞基因转移方法导入与脂质代谢及转运相关的基因,对由遗传因素引起的脂蛋白代谢紊乱达到有效的调节,并对阻止或逆转动脉粥样硬化的发生与发展也有重要意义。     

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。