摘要
目的研究逆转录病毒介导诱导型一氧化氮合酶(iNOS)基因在体外培养的大鼠主动脉平滑肌细胞(VSMC)中的表达。方法通过脂质体介导的DNA转染法将PLXSNiNOS导入PA317细胞中;经G418筛选获得抗性克隆;用NIH3T3细胞测定病毒上清滴度;将病毒上清转染体外培养的VSMC;采用RT-PCR、Westernblot检测VSMC内iNOS mRNA和蛋白的表达。结果PLXSNiNOS转染体外培养的VSMC 48 h后,在VSMC内可检测到外源性iNOS mRNA和蛋白;而用PLXSN转染体外培养的VSMC 48 h后未能检测到外源性iNOSmRNA和蛋白表达。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iNOS蛋白。
Objective To study the expression of inducible nitric oxide synthase（iNOS） gene mediated by retroviral vector in cultured aortic vascular smooth muscle cell（VSMC） of rat. Methods Recombinant retroviral vector PLXSN iNOS 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 viral supernatant. The expression of the retroviral iNOS transgene was examined by RT-PCR and Western blot analyses. Results mRNA and protein of transferred iNOS gene were detected 48 hours after gene transfer within the transfected cells. Alone PLXSN-treated VSMC showed no evidence of iNOS mRNA and protein. Conclusion iNOS gene could be quickly and effectively transferred into cultured VSMC by retroviral vector.
出处
《福建医科大学学报》
2006年第4期327-329,333,共4页
Journal of Fujian Medical University
基金
福建省自然科学基金资助项目(C0310023)
福建省科技厅重点课题资助项目(2004Y017)
福建省卫生厅青年基金资助项目(2004-1-15)
