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目的:研究HIV-1Nef基因对内皮细胞ECV304细胞ICAM-1表达的影响及其信号途径。方法:选用Nef基因稳定表达细胞株ECV304-Nef和对照细胞株ECV304pcDNA3.1(+),应用Westernblot分析ECV304-Nef细胞ERK的磷酸化水平,利用ERK磷酸化抑制剂通过Westernblot、流式细胞术分析ECV304-Nef细胞ICAM-1的表达水平与信号分子ERK磷酸化的相关性。结果:Westernblot显示ECV304-Nef细胞ICAM-1和p-ERK蛋白的表达水平均高于对照组;流式细胞术检测结果表明ECV304-Nef细胞和对照组ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P<0.01)。加入p-ERK抑制剂PD98059后,ECV304-Nef细胞p-ERK水平被显著抑制,ICAM-1降至对照组水平,ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(11.4±1.1)%和(10.4±1.5)%(P>0.05)。结论:HIV-1Nef基因上调血管内皮细胞细胞黏附分子ICAM-1的表达与ERK信号分子磷酸化有关,为HIV-1感染的致病机制及临床治疗提供实验基础。
Objective: To investigate the effect of HIV-1 Nef gene on the expression of ICAM-1 in ECV304 cells and its signal pathway. Methods: Nef gene-stable cell line ECV304-Nef and control cell line ECV304pcDNA3.1 (+) were selected. The phosphorylation level of ERK in ECV304-Nef cells was analyzed by Western blot. The ERK phosphorylation inhibitor was analyzed by Western blot and flow cytometry Correlation of ICAM-1 expression level with ERK phosphorylation in ECV304-Nef cells. Results: Western blot showed that the expression of ICAM-1 and p-ERK protein in ECV304-Nef cells were higher than that in control group. The results of flow cytometry showed that the percentage of ICAM-1 positive cells in ECV304-Nef cells and control group were (35.3 ± 2.2% and 12.5 ± 0.8%, respectively (P <0.01). After p-ERK inhibitor PD98059 was added, the level of p-ERK in ECV304-Nef cells was significantly inhibited and ICAM-1 was decreased to the level of control group. The percentage of ICAM-1 positive cells in ECV304-Nef cells and control cells were (11.4 ± 1.1) )% And (10.4 ± 1.5)%, respectively (P> 0.05). CONCLUSION: The up-regulation of ICAM-1 expression by the HIV-1 Nef gene is associated with the phosphorylation of ERK signaling molecules, providing the experimental basis for the pathogenesis and clinical treatment of HIV-1 infection.