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目的研究HIV-1的调节基因Nef对ECV304细胞ICAM-1表达的影响,从而为分析HIV-1感染引起内皮细胞生物学活性的变化,以及为阐明Nef参与HIV-1致病的分子机制奠定基础。方法应用本实验室已经建立保存的HIV-1Nef基因在内皮细胞的稳定表达细胞株ECV304-Nef和其阴性对照细胞株ECV304 pcDNA 3.1(+),通过RT-PCR、实时定量PCR(real-time PCR)、Western blot、FCM和细胞黏附试验分析ECV304-Nef细胞ICAM-1的表达水平。结果 RT-PCR、real-time PCR结果显示ECV304-Nef细胞ICAM-1 mRNA表达水平明显升高,为对照组的(4.3±0.2)倍;Western blot结果示ECV304-Nef细胞I-CAM-1蛋白的表达水平高于对照组;FCM分析显示ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P<0.01),两组间ICAM-1表达有显著差异。细胞黏附实验观察到ECV304-Nef细胞黏附的Jurkat细胞数明显多于对照组,荧光仪定量分析结果显示ECV304-Nef细胞黏附的Jurkat细胞的荧光强度值显著高与对照组(P<0.05)。结论本实验证实了HIV-1Nef基因可以上调血管内皮细胞细胞黏附分子ICAM-1的表达。
Objective To investigate the effect of HIV-1 regulatory gene Nef on the expression of ICAM-1 in ECV304 cells so as to provide a basis for the analysis of changes in endothelial cell biology activity induced by HIV-1 infection and to elucidate the molecular mechanism of Nef’s involvement in the pathogenesis of HIV-1 . METHODS: The stable expression cell line ECV304-Nef and the negative control cell line ECV304 pcDNA 3.1 (+) of HIV-1 Nef gene in endothelial cells have been established in our laboratory. Real-time PCR ), Western blot, FCM and cell adhesion assay were used to analyze the expression of ICAM-1 in ECV304-Nef cells. Results The results of RT-PCR and real-time PCR showed that the expression level of ICAM-1 mRNA in ECV304-Nef cells was significantly higher than that in control group (4.3 ± 0.2) FCM analysis showed that the percentage of ICAM-1 positive cells in ECV304-Nef cells and control cells were (35.3 ± 2.2)% and (12.5 ± 0.8)%, respectively (P <0.01) -1 expression was significantly different. The number of Jurkat cells adhered to ECV304-Nef cells was significantly higher than that of control cells in cell adhesion assay. The results of fluorimetric analysis showed that the fluorescence intensity of Jurkat cells adhered to ECV304-Nef cells was significantly higher than that of control cells (P <0.05). Conclusion This experiment confirmed that HIV-1 Nef gene can up-regulate the expression of ICAM-1 in vascular endothelial cells.