Resistin基因对TNF-α诱导血管内皮细胞ICAM-1表达的影响

Effects of resistin gene on TNF-α-induced expression of ICAM-1 in vascular endothelial cells

目的:研究抵抗素(resistin)基因对TNF-α诱导血管内皮细胞ECV304表达细胞间黏附分子1(ICAM-1)的影响。方法:应用分子生物学技术,将resistin基因克隆到载体pEGFP-C1中构建质粒pEG/Resi,脂质体转染ECV304,转染6h加入20μg/LTNF-α诱导,用RT-PCR方法分别检测转染18、30、42和54h时resistin和ICAM-1 mRNA表达;同时用免疫印迹法检测resistin蛋白的表达,并用ELISA方法检测各期ECV304上清ICAM-1蛋白表达。结果:经酶切鉴定和测序证实质粒pEG/Resi构建成功并可在ECV304中表达;相同TNF-α诱导条件下转染resistin基因组的ICAM-1表达水平始终高于未转染组,在resistin基因表达高峰(30和42h)ICAM-1 mRNA和蛋白表达量较未转染组均显著升高(P<0.05);且ICAM-1 mRNA和蛋白表达水平均随resistin基因表达量的增加而升高。结论:Resistin基因能够增强TNF-α诱导ECV304细胞表达ICAM-1。

Objective：To study the effect of resistin gene on TNF-α-induced expression of ICAM-1 in ECV304 cells. Methods： Resistin gene was cloned into vector pEGFP-C1 to construct pEG/Resi plasmid; the latter was then used to transfect ECV304 cells via liposome. TNF-α（20μg/L） was added to the system 6 h after transfection. RT-PCR was used to detect the expression of resistin and ICAM-1 mRNA in the cells 18, 30, 42 and 54 h after transfection; meanwhile, the resistin protein levels were determined by Western blotting analysis. ICAM-1 protein levels in the supernatants of ECV304 cells were determined by ELISA. Results： Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was successfully constructed and was expressed in ECV304. ICAM-1 levels in resistin-transfected group were consistently higher than that in the non-transfected group in the presence of TNF-α. ICAM-1 mRNA and protein expression in the transfected-group was significantly higher than that in the non-transfected group during the peak periods （30 and 42 h） of resistin expression （P〈0.05） ; ICAM-1 mRNA and protein expression increased with the increase of resistin gene expression. Conclusion： Resistin gene can enhance TNF-α-induced expression of ICAM-1 in ECV304 cells.