稳定表达组织型纤溶酶原激活因子基因的人脐静脉内皮细胞株的建立  

The Stable Expression of Human Tissue-type Plasminogen Activator Gene Mediated by Lipofectamine in Human Vein Endothelial Cell Line Cells

作　　者：刘小斌[1] 张凯伦[2] 蒋雄刚[2] 王家宁[3] 黄永章[3] 

机构地区：[1]贵州省人民医院心外科,贵阳550002 [2]华中科技大学同济医学院附属协和医院心血管外科,武汉430022 [3]湖北郧阳医学院临床医学研究所,十堰442000

出　　处：《生物医学工程学杂志》2007年第6期1330-1333,共4页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30571838)

摘　　要：为建立稳定表达组织型纤溶酶原激活因子(t-PA)基因的人脐静脉内皮细胞株,为其在基因修饰的组织工程血管中的应用奠定基础。首先构建t-PA基因的真核表达载体pcDNA3.1-Myc-HisB(-)/t-PA,将该表达载体用阳离子脂质体介导,转染人脐静脉内皮细胞系细胞,经G418筛选,获得阳性细胞克隆,扩增后分别用逆转录聚合酶链反应(RT-PCR)、免疫印迹(Western-blotting)检测t-PA转录和蛋白表达水平,用底物发色法检测t-PA的活性。逆转录聚合酶链反应检测出了t-PA的稳定转染细胞克隆,免疫印迹证实该克隆细胞有含Myc标签的t-PA蛋白表达,底物发色法检测发现其表达产物的活性增加。结果证明成功建立了稳定表达t-PA基因的人脐静脉内皮细胞株。We have established a human umbilical vein endothelial cell （HUVEC） line monoclonal cells with the stable expression of human tissue-type plasminogen activator （t-PA） gene to provide a basis for further study on the vascular tissue engineering. Recombinant plasmid pcDNA3. 1-Myc-His B （-）/t-PA was constructed by insertion of t-PAcDNA originated from PBS/t-PA into eukaryotlc expression vector pcDNA3.1-Myc-His B（-） and transfected into hUVEC llne cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The transcription and expression of t-PA gene were investigated by RT-PCR and Western blotting respectively. The t-PA activity was measured by chromogenic substrate assay. The positive clone cells which transcripted the mRNA of t-PA gene was obtained by RT-PCR. Immunoreactive human t-PA of the medium was significantly increased in the group of transfected gene when compared with that in the controlled and transfected plasmid without t-PA gene group. The biological activity of the protein of the t-PA in the media was increased significantly in the positive clone cells with t-PA gene transfected. The HUVEC llne monoclonal cells with the stable expression of t-PA gene was established successfully.

关 键 词：血管组织工程 组织型纤溶酶原激活因子 阳离子脂质体 稳定表达 

分 类 号：R346[医药卫生—基础医学]