内皮型一氧化氮合酶与血管结构及功能的相关性

Correlation of endothelial nitric oxide synthase with vascular structure and function

目的:应用内部核糖体进入位点构建携带内皮型一氧化氮合酶和血管内皮生长因子的双基因表达载体,研究外源性基因内皮型一氧化氮合酶和血管内皮生长因子在人脐静脉内皮细胞ECV304中的转染表达,探讨内皮型一氧化氮合酶与血管结构及功能的相关性。方法:实验于2003-12/2004-12在南京鼓楼医院科研部完成。将Phcmvsp1a-enos中内皮型一氧化氮合酶基因和PcDNA-vegf中血管内皮生长因子基因定向克隆到真核表达载体PcDNA-ires中,构建重组质粒PcDNA-enos-ires-vegf,经酶切,聚合酶链反应扩增和部分DNA测序分析证实后,转染人脐静脉内皮细胞,硝酸还原酶法检测转染后细胞中一氧化氮和一氧化氮合酶活性,免疫组化和荧光双标记及westernblotting检测转染后细胞蛋白水平的表达,Trizol提取总RNA,反转录-聚合酶链反应测定转染后细胞mRNA水平的表达。结果:成功构建携带内皮型一氧化氮合酶和血管内皮生长因子的双基因表达载体PcDNA-enos-ires-vegf,双基因内皮型一氧化氮合酶和血管内皮生长因子在人脐静脉内皮细胞基因组中获得整合及表达。重组基因PcDNA-enos-ires-vegf组与对照组PcDNA-enos在一氧化氮含量和一氧化氮合成酶活性的表达差异无显著性意义。PcDNA-enos-ires-vegf转染细胞后转染效率为30%～40%。

AIM:To construct the double gene expression vector of endothelial nitric oxide synthase (enos) and vascular endothelial growth factor (vegf) by internal ribosome entry sites (ires) sequences, study the transfection and expression of endothelial nitric oxide synthase (ENOS) and vascular endothelium growth factor (VEGF) on human umbilical vein endothelial ECV304 cells, and investigate the correlation of ENOS with vascular structure and function. METHODS:The experiment was carried out in the scientific research department of Nanjing Gulou Hospital from December 2003 to December 2004. Enos in Phcmvsp1a-enos and vegf in PcDNA-vegf were directionally cloned into eukaryotic expression vector PcDNA-ires to construct recombinant plasmid PcDNA-enos-ires-vegf.After constructed and identified by restriction endonuclease analysis, polymerase chain reaction (PCR) amplification and partial DNA sequence analysis,PcDNA-enos-ires-vegf was transfected into ECV304 cell, and then content of nitric oxide (NO) and activity of nitric oxide synthase (NOS) were determined with the method of nitrate reductase, protein expression was assayed with immunohistochemistry,immunofluorescence and western blotting, and mRNA expression was detected with by reverse transcription-PCR (RT-PCR) after total RNA was extracted with Trizol. RESULTS:The PcDNA-enos-ires-vegf double gene expression vector of enos and vegf was successfully constructed.Double gene of enos and vegf was successfully co-transfected and expressed on ECV304 cells.The expressions of NO content and NOS activity were not significant between the recombinant gene PcDNA-enos-ires-vegf group and PcDNA-enos control group.The transfected efficiency after PcDNA-enos-ires-vegf transfection was 30%to 40%. CONCLUSION: The successful construction of double gene expression vector PcDNA-enos-ires-vegf and the expression of double gene enos and vegf establish foundation for investigating the role of ENOS in vascular structure and function.