构建内皮型一氧化氮合酶真核表达载体及在内皮细胞的表达  

Endothelial nitric oxide synthase expression in endothelial cells following aukaryotic expression vector construction

作　　者：乔彤[1] 刘长建[1] 冉峰[1] 周敏[1] 乔威[1] 张乐[1] 李雷[1] 

机构地区：[1]南京大学医学院附属鼓楼医院血管外科,江苏省南京市210008

出　　处：《中国组织工程研究与临床康复》2008年第46期9080-9084,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:内皮型一氧化氮合酶表达产物对内皮细胞的影响是血管外科基础研究中的重要课题,对于生物人工血管的研制有重要意义。目的:构建内皮型一氧化氮合酶基因真核表达载体pcDNA3.1/eNOS,以脂质体为介导观察其在血管内皮细胞中的转染效率,评价表达产物内皮型一氧化氮合酶的酶学活性及其对内皮细胞生长的影响。设计、时间及地点:单一样本观察,实验于2005-07/2007-05在南京大学医学院附属鼓楼医院生物化学实验室完成。材料:质粒PCMV-eNOS由勋阳医学院张群林教授惠赠。pcDNA3.1真核表达载体由南京大学博士后贾立军馈赠,pcDNA3.0/EGFP质粒由戚金亮博士提供。大肠杆菌DH5a和人脐静脉内皮细胞株(ECV304)为南京大学生物系保存。方法:采用分子生物学技术,先用限制性核酸内切酶EcorⅠ酶切质粒PCMV/eNOS,电泳回收3.6kb编码人内皮型一氧化氮合酶cDNA序列,克隆入质粒pcDNA3.1的EcorⅠ酶切位点,通过内皮型一氧化氮合酶cDNA序列中的XhoⅠ酶切位点筛选其插入载体的方向,构建pcDNA3.1/eNOS重组质粒。以Lipofec-tamine为介导将其与pcDNA3.0/EGFP共转染分离培养的人脐静脉内皮细胞(ECV304)。主要观察指标:①观察转染后对ECV304生长的影响,在流式细胞仪和荧光显微镜下计算转染效率。②以反转录-聚合酶链反应和免疫组织化学检测内皮型一氧化氮合酶基因的表达,测定内皮型一氧化氮合酶活性以及一氧化氮产量。③观察施加不同因素(L-精氨酸、氯化钙、乙二胺四乙酸、L-NAME)对内皮型一氧化氮合酶活性及一氧化氮合成的影响;同时检测此时ECV304生长所受影响。结果:以脂质体为介导转染人脐静脉ECV304后,其转染效率为(32.6±2.6)%,反转录-聚合酶链反应和免疫组织化学检测到相应的内皮型一氧化氮合酶mRNA和蛋白表达,与对照组有明显差别;同时在上述不同因素影响下内皮型一氧化氮合酶活性出�BACKGROUND： It is one of key subjects to evaluate the the effect of endothelial nitric oxide synthase （eNOS） activity on endothelial cell culture, which has important significance for preparing tissue engineered vascular grafts. OBJECTIVE： To construct the eukaryotic expression vector pcDNA3.1/eNOS for detecting the transfection efficiency of eNOS in vascular endothelial cells and to evaluate the effect of eNOS activity on endothelial cell culture. DESIGN, TIME AND SETTING： The single sample observation was performed at the Labboratory of Biochemistry, Affiliated Drum Tower Hospital of Nanjing University Medical School from July 2005 to May 2007. MATERIALS： Plasmid PCMV-eNOS was presented by Zhang Qun-lin, a professor from Yunyang Medical College. Eukaryotic expression vector pcDNA3.1 was presented by Jia Li-jun, a post-doctor from Nanjing University. The pcDNA3.0/EGFP was provided by Doctor Qi Iin-liang and DH5a and ECV304 were conserved by the Biology Department of Nanjing University. METHODS： The full-length human eNOS cDNA（3.6kb） was isolated from pCMV/eNOS by restriction enzyme digest with EcorI, and then ligated into the EcorI cloning site of the pcDNA3,1 expression plasmid and judged the eNOS direction by restriction enzynme digest with Xho Ⅰ to construct recombint pcDNA3.1/eNOS. Co-transfection of pcDNA3.1/eNOS with pcDNA3.0/EGFP mediated by Lipofectamine into human umbilical vein endothelial cells （ECV304） was performed. MAIN OUTCOME MEASURES： Transfection rate and effect of ECV304 proliferation were calculated by fluorescence microscope and flow cytometer. RT-PCR and immunocytochemistry were used to detect the status of eNOS expression. When treated ECV304 with four independent factors including Ca2＋, L-arginine（L-Arg）, Ethylene Diamine Tetraacetic Acid （EDTA） and N-nitro-L-arginine methylester （L-NAME）, the eNOS activity and nitric oxide release were detected. Meanwhile, changes in the ECV304 proliferation were examined. RESULTS： After transfected liposome into

关 键 词：内皮型一氧化氮合酶 真核表达载体 人脐静脉内皮细胞 

分 类 号：R512.6[医药卫生—内科学] R544.1[医药卫生—临床医学]