逆转录病毒介导诱导型一氧化氮合酶基因转染抑制血管平滑肌细胞增殖的研究

目的：研究逆转录病毒介导诱导型一氧化氮合酶（iNOS）基因转染对体外培养的大鼠主动脉血管平滑肌细胞（VSMC）增殖的影响,探讨iNOS转基因治疗血管移植术后再狭窄的可行性。方法：将不同滴度的病毒上清转染体外培养的VSMC;采用RT-PCR、Western-blot检测VSMC内iNOSmRNA和iNOS蛋白的表达;用Griess法检测iNOS转基因细胞的培养液中一氧化氮（NO）的含量;用改良MTT、法检测iNOS转基因对VSMC增殖的抑制作用。结果：不同滴度的PLXSNiNOS转染体外培养的VSMC48h后,在VSMC内可检测到外源性iNOSmRNA和iNOS蛋白,表达水平随病毒滴度的增加而增强,呈现剂量依赖性;而用最高滴度的PIXSN转染体外培养的VSMC48h后,在VSMC内未能检测到外源性iNOSmRNA和iNOS蛋白表达;iNOS转基因细胞的培养液中NO含量显著增高,同时VSMC增殖受到明显抑制,均呈现剂量依赖性。结论：逆转录病毒介导iNOS基因可高效转染体外培养的VSMC,并在细胞内表达活性的iNOS蛋白,而且产生大量的NO,明显抑制VSMC增殖。为iNOS转基因治疗血管移植术后再狭窄的临床应用提供有力的实验依据。

Inhibition of retrovirus-mediated gene transfer of inducible nitric oxide synthase on proliferation of vascular smooth muscle cell

Aim： 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） of rat and the possibility of iNOS gene therapy for vessel graft restenosis. Methods： 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 analysis. 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 MTr assay. Results： mRNA and protein of transferred iNOS gene were detected 48 hours postgene transfer within the transfected cells. Levels of iNOSmRNA and protein in PIXSN-iNOS infected cells were positively correlated with viral titer of viral supematant. PLXSN-treated VSMC showed no evidence of iNOS mRNA and protein. 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. Conclusion： 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.