Inhibition Of Retrovirus-mediated Gene Transfer Of Inducible Nitric Oxide Synthase On Proliferation Of Vascular Smooth Muscle Cell

Objective To study the effect of inducible nitric oxide synthase(iNOS) gene mediated by retroviral vector on the proliferation of cultured aortic vascular smooth muscle cell(VSMC) and the possibility of iNOS gene therapy for vessel graft restenosis.Methods Recombinant retroviral vector PLXSN-iNOS or PLXSN was transferred into the amphotropic packaging cell line PA317 by means of lipofectamine-mediated method. Anti-G418 positive clones were selected by G418. The viral titer was determined with the NIH3T3. Ex vitro VSMC were transfected by different viral titer of viral supernatant. The expression of the retroviral iNOS transgene was examined by RT-PCR and Western blot analyses; Nitric oxide(NO) release from infected cells was determined by Griess reaction; The inhibition of iNOS transgenosis on the proliferation of VSMC was detected by modified MTT assay.Results 1. The titer of recombinant retroviral vector PLXSN-iNOS and PLXSN was 8.7×105CFU/ml and 8.1×105CFU/ml, respectively. 2. mRNA and protein of transferred iNOS gene were detected 48 hours post-gene transfer within the transfected cells. Levels of iNOSmRNA and protein in PLXSN-iNOS infected cells were positively correlated with viral titer of viral supernatant. PLXSN-treated VSMC showed no evidence of iNOS mRNA and protein. 3. Transfection of PLXSN-iNOS into cultured VSMC resulted in a dose-dependent increase in NO production. And iNOS transgenosis significantly inhibited proliferation of VSMC. The inhibition effect was positively correlated with viral titer of viral supernatant. Conclusions iNOS gene could be quickly and effectively transferred into cultured VSMC by retroviral vector and its expression could significantly inhibit the proliferation of cultured VSMC. Retrovirus-mediated gene transfer of iNOS might play an important role in prevention of restenosis.