| 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 VSMC 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 cDNA (3.96kb) was isolated from pSPORT-iNOS by restriction enzyme digested with Hind III and Xbl I, and was then ligated into the Hind III /Xbl I cloning site of the pcDNA3 expression plasmid. pcDNA3-iNOS, constructed the eukaryotic expression vector, delivered into cultured VSMC by lipofect DOTAP liposomal. The gene expression of iNOS mRNA was quantified by reverse transcription-polymerase chain reaction (RT-PCR). iNOS expression wasobserved by Western blot and immunofluorescence. The antibody used specifically recognizes the iNOS isoform (131kDa) and does not cross-react with the endothelial forms of NOS. NO product in the infected cells was determined by measuring nitrite (NO2-) release using the Griess reaction. 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 cytomegalovirus 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 VSMC cells infected pcDNA3-iNOS was obtained iNOS band at 462bp by RT-PCR, no iNOS band was found in the other group. It indicates that the cells infected with pcDNA3-iNOS can express iNOS mRNA.After iNOS gene transfected to VSMC, 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 NO2- in the supernatants was quantified with the Griess reaction using sodium nitrite. The results showed:the content of NO in pcDNA3 cells was minimal, with NO2- production (87±12.51) just above the normal group (68.29±13.48),(P=0.1,n=6). NO2- production by pcDNA3-iNOS cells was higher (225.87±27.68) (n=6, P=0.00 vs. the normal cells or pcDNA cells).The effect of NO on cell cycle distribution of VSMC was analyzed by FCM. Under control conditions, 61.13±0.93% of cells in G0/G1 phase, 26.47±0.67% were in S phase, and 12.4±0.61% were in G2/M phase,added supernatants pcDNA3 cells, the cell cycle was slightly different with the control cells (P=0.46, n=3). After treatment with pcDNA3-iNOS cells supernatants for 48h, 86.6±0.68% of cells in G0/G1 phase, 9.34±0.59% were in S phase, 6.68±0.12% were in G2/M phase. Compared with the control cells, the number of cells in G0/G1 phase was much higher (P=0.034, n=3), but the number in S phase was decreased (P=0.026, n=3), the number in G2/M phase was a little different (P=0.73, n=3).Conclusion: 1. The eukaryotic expression vector, pcDNA3-iNOS, was...
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