人脂联素基因真核表达重组体的构建与在体表达.docVIP

  人脂联素基因真核表达重组体的构建与在 体表达# 杨文凯，陈新忠，夏东升** 5 10 15 20 25 （华中科技大学同济医学院附属协和医院心血管外科） 摘要：目的：构建含人脂联素基因 apMl 的真核表达载体，采用超声微泡声诺维介导 apMl 基因转染并表达于兔主动脉血管，为脂联素用于体内干预动脉粥样硬化的研究奠定基础。方 法：自人心外膜脂肪组织提取总 RNA，RT-PCR 扩增 apMl 片段，克隆入载体 pcDNA3.1 中， 形成 pcDNA3.1-apMl 重组体，经限制性核酸内切酶酶切鉴定后对其进行测序。经兔耳缘静 脉注射 pcDNA3.1-apMl 与声诺维的混合物，同时采用诊断超声仪照射兔胸腹主动脉处 3 分 钟，分别于照射后 2 天、7 天、14 天取兔主动脉血管及血清，Western Blot 检测兔主动脉中 apMl 基因的表达，ELISA 检测兔血清中 apMl 蛋白含量。结果：重组质粒 pcDNA3.1-apMl 经测序鉴定正确，Western Blot 检测显示声诺维传输 apMl 基因 2 天后即可检测到 apMl 基因 高表达，7 天后仍显示高表达，表达可持续至 14 天。ELISA 检测显示声诺维传输 apMl 基因 可显著增加兔血清 apMl 蛋白含量（p0.01）。结论：成功制备了 apMl 基因真核表达重组体， 声诺维可在快速血流状态下，安全、高效传输 apMl 基因入兔主动脉血管壁并有效表达与分 泌，建立了一种新的 apMl 基因在体传输技术，为脂联素进一步用于体内干预动脉粥样硬化 的研究提供了新的技术方法。 关键词：apMl；声诺维；动脉粥样硬化 中图分类号：R 589.2 Construction and expresssion in vivo of recombinant plasmid containing human apM1 gene YANG Wenkai, XIN Zhongchen, XIA Dongsheng (Department of cardiovascular Surgery,Union Hospital,Tongji Medical college,Huazhong University of Science and Techology,WuHan 430022) Abstract: Objective To construct recombinant plasmid containing human apMl gene and delivery apMl gene into aorta of rabbits by using ultrasound with SonoVue for the gene therapy 30 35 40 research on atherosclerosis. Methods apMl mRNA from epicardial fat was amplified by RT-PCR and cloned into plasmid pcDNA3.1 to generate the recombinant plasmid pcDNA3.1-apMl, which was confirmed by nucleotide sequencing. While the mixture of SonoVue and pcDNA3.1-apMl was injected though helix vein, the thoracoabdominal aorta of rabbits were sonicated by ultrasound for 3min. The arterial vessels and blood serum of rabbits were harvested at 2, 7and 14 days respectively after delivery to detect apMl expression by Western blot analyses and ELISA. Results The identification of pcDNA3.1-apMl recombinant by DNA sequencing is correct. apMl gene could be effectively transfered into the aorta wall of rabbit by using ultrasound with SonoVue. The high expression of apMl gene was detected at 2 days and 7 days after d