| Background: Nitric oxide (NO) is a potent vasodilator generated by endothelium. To be an important messenger molecule, NO involves in diverse processes in many tissues and has been shown to serve many vasoprotective roles in cardiovascular systems: promoting reendothelialization, preventing platelet and leukocyte adherence, inhibiting vascular smooth muscle cell (VSMC) proliferation and migration. These functions preserve a normal vascular environment. Endothelial NO production is dysfunctional after arterial injury, such as atherosclerosis, hypertension and restenosis. Restoration of NO production in vessels plays an important role in therapy of these diseases. A number of studies have established nitric oxide synthase (NOS) gene transfer in luminal delivery, which enhanced endotheliaum-dependent relaxation. The goal of the present study was to determine whether fibroblast V79 could be transduced with vector-encoding human inducible NOS (iNOS), result in functional expression and increase NO release. It can give assertive evidence for introducing the gene vector in adventitial delivery in vivo. Methods: The full-length human iNOS eDNA (3.96kb) was isolated from pSPORT-iNOS by restriction enzyme digest with Hind III and Xbl I, and was then ligated into the Hind III IXbl I cloning site of the pcDNA3 expression plasmid. pcDNA3-iNOS, constructed the eukaryotic expression vector, delivered into cultured fibroblast V79 by lipofect AMINE. The clone cells were * The study was supported by the grand from Natural Scientific Fundation of China (NSFC, No 39970276). selected by ~ The gene expression of iNOS mRNA was quantified by reverse transcription-polymerase chain reaction (RT-PCR). iNOS expression was observed by Western blot and immunofluorescence. The antibody used specifically recognizes the iNOS isoform (13 lkDa) and does not cross-react with the endothelial forms of NOS. NO product in the infected cells was determined by measuring nitrite (N02) release using the Griess reaction. The cell medium was replaced with medium alone or medium with H4B (1OOp~M), and the effect of 114B addition on NO synthesis was also observed. Added the transferred cell supernatants, the influence on VSMC proliferation induced by NO was studied. The VSMC cycle was analyzed with flow cytometry (FCM). Results: The mammalial expression vector, pcDNA3-iNOS, contains a cytomegaloverus promoter and the cDNA for recombinant human iNOS. It has a neo (neomycin phosphotransferase) gene to allow selection of the transformants in the presence of G418 after transfection. iNOS gene expression was assessed by RT-PCR in normal cell group~ pcDNA3-transfected group and pcDNA3-iNOS group. It showed that V79 cells infected pcDNA3-iNOS was obtained iNOS band at 462bp by RT-PCR, no iNQS band was found in the other group. It indicates that the cells infected with pcDNA3-iNOS can express iNOS mRNA. At 2 weeks after iNOS gene transfection by 0418 screening, significant levels of iNOS protein expression were detected by Western blot, the iNOS protein in the cytochylema was showed by immunofluorescence, but the normal group and control vector group cells were not found iNOS protein expression by Western blot and immunofluorescence. Accumulated N02 in the supernatants was quantified with the Griess reaction using sodium nitrite. The results showed: medium without H4B, the content of NO in pcDNA3 cells was minimal, wi... |
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