真核表达载体pCMV-(Kozak)TFPI在搭桥模型移植静脉中的表达  

Expression of eukaryotic expressed plasmid vector pCMV-(Kozak)TFPI in vein grafts of bypass graft model

作　　者：厉泉[1] 张凯伦[1] 蒋雄刚[1] 孙图成[1] 龚永生[1] 祖育昆[1] 郭超[1] 徐鹏[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心外科,湖北武汉430022

出　　处：《中国老年学杂志》2008年第7期628-630,共3页Chinese Journal of Gerontology

基　　金：国家自然科学基金资助项目(No30571838)

摘　　要：目的构建真核表达载体pCMV-(Kozak)TFPI并检测其在移植静脉中的表达,为冠状动脉旁路移植术中转染大隐静脉内皮细胞抗凝治疗的临床试验奠定理论应用基础。方法用逆转录聚合酶链反应(RT-PCR)的方法提取人组织因子途径抑制因子(TFPI)基因,并引入Kozak序列,将(Kozak)TFPI亚克隆入pCMV质粒中。在阳离子脂质体介导下,转染颈总动脉搭桥模型移植静脉内皮细胞。RT-PCR、蛋白印迹法(Western印迹)和免疫组化法检测移植静脉中外源TFPI基因mRNA和蛋白的表达。结果(Kozak)TFPI成功克隆入pCMV中。RT-PCR、Western印迹和免疫组化检测到移植静脉中外源TFPI基因mRNA和蛋白的表达。结论pCMV-(Kozak)TFPI真核表达载体构建成功,移植静脉中检测到其蛋白的表达。Objective ： To provide the theoretical basis for future clinical trials of anticoagulation therapy about tissue factor pathway inhibitor （TFPI） gene transfected into saphenous vein grafts in coronary artery bypass grafting by constructing eukaryotic expressed plasmid vector pCMV-（ Kozak）TFPI and detecting its expression in vein grafts. Methods The full length human TFPI gene extracted by RT-PCR was introduced with Kozak sequence. （ Kozak）TFPI sequence was inserted in pCMV. Vein graft endotheliocytes of common carotid artery bypass graft model were transfectcd with cationic liposome. RT-PCR, Western blot and immunohistochemistry were used to detect expression of exogenous TFPI mRNA and protein in vein grafts. Results The （ Kozak）TFPI sequence was successfully cloned into pCMV. RT-PCR,Western blot and immunohistochemistry results showed the expression of exogenous TFPI mRNA and protein in vein grafts. Conclusions The eukaryotic expressed plasmid vector pCMV-（Kozak） TFPI is constructed successfully,and TFPI protein expression is detected in vein grafts.

关 键 词：冠状动脉旁路移植术 组织因子途径抑制因子 移植静脉 转染 

分 类 号：R654.2[医药卫生—外科学] R541.4[医药卫生—临床医学]