重组人诱导型一氧化氮合酶在V_(79)细胞中的表达及四氢叶酸对其活性的影响

Expression of human inducible nitric oxide synthase in fibroblast cell line V_(79) and effects of H_(4)Bon iNOS activity

目的 :探讨重组人诱导型一氧化氮合酶 (iNOS)基因在V79成纤维细胞中转染的可行性及四氢叶酸(H4 B)对转染后产生NO的影响。方法 :将人iNOS基因通过真核表达载体转入V79细胞中 ,经G4 1 8筛选 ,RT -PCR、免疫荧光法进行鉴定。采用Griess法测定加入H4 B前后NO- 2 的生成量。结果 :iNOS基因转染组V79细胞中有iNOSmR NA的表达 ,细胞胞质中可见分泌的iNOS蛋白。未加H4 B时 ,iNOS转染组NO- 2 含量明显高于正常细胞组和空质粒转染组 (P <0 0 1 ,n =6) ,正常细胞组和空质粒转染组之间NO- 2 含量无明显差异 (P >0 0 5 ,n =6) ;加入H4 B后 ,iNOS转染组中NO- 2 含量较未加组明显增加 ,两者相比具有显著差异 (P <0 0 1 ,n =6) ,而正常细胞组和空质粒转染组与未加H4 B组相比无明显变化 (P >0 0 5 ,n=6)。结论 :通过外界加入H4 B能显著增加成纤维细胞转染产生的iNOS的活性 。

AIM: To explore the feasibility of human iNOS transfected into V 79 cells by gene transfer and investigate the effects of H 4B on iNOS activity. METHODS: Human iNOS was transfected into V 79 cells with the karyocyte expressive vector. The cloned cells were selected by G 418 . The expression of iNOS mRNA was quantified by RT-PCR and iNOS expression was observed by immunofluorescence. NO product in cells was determined by measuring nitrite (NO - 2) release using the Griess reaction. RESULTS: V 79 cells infected human iNOS was proved to have iNOS mRNA at 462 bp by RT-PCR, and iNOS protein in the cytochylema by immunofluorescence. When the cells were incubated without H 4B, the content of NO in pcDNA 3 cells was minimal, with NO - 2 production (82.32±13.08) just above the normal group (74 38±9 80, P> 0 05, n= 6) There was no significant difference between pcDNA 3 cells incubated with or without H 4B, ( P> 0.05, n= 6) NO - 2 production by pcDNA 3-iNOS cells without H 4B was higher (105 58±13 33) ( n= 6, P< 0 01 vs the normal cells or pcDNA cells). However, in pcDNA 3-iNOS cells incubated with H 4B, NO - 2 production was much higher (236 57±31 83) ( n= 6, P< 0 01 vs the all former groups). CONCLUSION: iNOS activity was increased by adding H 4B in pcDNA 3-iNOS cells, and the fibroblast can be a target cell of iNOS gene transfer.