重组人诱导型一氧化氮合酶在V_(79)成纤维细胞株中的表达及意义探讨

Expression and significance of recombinant human inducible nitric oxide synthase gene in fibroblast cell line V_(79) in vitro

目的 观察人诱导型一氧化氮合酶 (iNOS)基因转染至V79成纤维细胞株后 ,iNOS的表达和分泌一氧化氮的情况 ,以探讨血管壁成纤维细胞分泌一氧化氮的可行性。方法 构建真核表达载体pcDNA3 iNOS,将人iNOS基因转入V79细胞中 ,经G41 8筛选 ,克隆扩增 ,通过RT PCR、免疫荧光法进行鉴定。结果 pcDNA3 iNOS基因转染的V79成纤维细胞中有iNOSmRNA的转录 ,在细胞胞质中可见分泌的iNOS蛋白 ,iNOS转染组一氧化氮的含量 (2 37.67± 2 7.2 8)与正常细胞组 (78.2 6± 1 1 .77)和空质粒组 (84.2 2± 1 2 .67)相比有显著性意义 (P <0 .0 0 1 ) ,但正常细胞组和空质粒组相比无显著性差异 (P >0 .0 5)。结论 iNOS基因可以成功地转入成纤维细胞中并能使转染的成纤维细胞具有分泌NO的功能 。

Objective To observe the expression of nitric oxide synthase (NOS) and secretion of NO by fibroblast cell line V 79 into which human recombinant inducible NOS (iNOS) was transferred so as to explore the feasibility of NO release by fibroblasts via gene transfer. Methods The eukaryotic expression vector pcDNA 3 iNOS was constructed and then transfected into V 79 cells. After the screening with G 418 and cloning, the cells were verified with RT PCR and immunofluorescence staining. V 79 cells were cultured with the medium containing 100 μmol/L H4B and NO content in the supernatant was quantified with the Griess reaction by using sodium nitrite as standard. Results The expression of iNOS mRNA in V 79 cells transfected with pcDNA 3 iNOS was detected and the NO concentration in the culture supernatant of stable iNOS-expressing V 79 cells was much higher than control cells and the cells with pcDNA 3 plamid transfection, but the control and vector group showed no significant difference ( P >0.05). Conclusion Recombinant iNOS could be successfully transfected and expressed in fibroblasts after the eukaryotic expression vector transfection, and increase the NO concentration. This gene transfer is possible to substitute endothelial function of NO release.