摘要目的检测切应力诱导人脐静脉血管内皮细胞白介素-8(IL-8)基因表达,探讨其转录激活机制.方法选用4.2 dyne/cm2层流低切应力刺激细胞.采用RT-PCR检测切应力刺激后内皮细胞IL-8 mRNA表达情况.同时构建IL-8上游调控序列(IL-8 USCS)的绿色荧光增强蛋白报告基因pEGFP1-IL8USCS,通过转基因和流式细胞术检测切应力诱导IL-8基因的转录激活.用免疫荧光细胞化学染色观察NF-κB核转移情况.免疫印迹检测切应力诱导IκB的磷酸化和降解.用RT-PCR、Northern杂交和免疫荧光细胞化学染色在mRNA和蛋白水平检测Toll-样受体-4(TLR-4)的表达.结果用切应力刺激120 min后,内皮细胞表达IL-8 mRNA明显增强.切应力刺激180 min后pEGFP1-IL8USCS转染的血管内皮细胞绿色荧光蛋白表达显著增强.NF-κB 5免疫荧光细胞化学染色显示,切应力刺激30 min,胞核即出现阳性反应,刺激90 min后,胞核呈强阳性染色.切应力刺激10 min时磷酸化IκB即显著增强,60 min后磷酸化IκB印迹强度降到无,而IκB随刺激时间延长而逐渐降低.脐静脉血管内皮细胞表面表达Toll样受体-2(TLR-2)和TLR-4受体,当切应力刺激60 min后,TLR-4 mRNA表达明显增强.结论流体切应力刺激血管内皮细胞表达IL-8基因与NF-κB活化有关,天然免疫受体Toll可能介导这一反应过程.

abstractsObjective To examine interleukin-8 (IL-8) mRNA expression induced by flow shear stress in human umbilical vein endothelial cells (HUVECs) and investigate its transcriptional activation.Methods Flow laminar shear stress 4.2 dyne/cm2 was used for the stimulating experiments. The flow shear stress-induced IL-8 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). pEGFP1 was used to construct IL-8 reporter gene pEGFP1-IL8USCS for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-κB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IκB phosphorylation and degradation. RT-PCR, Northern blot, immunocytofluorescent staining and laser confocal microscopy were used to determine Toll-like receptor-4 (TLR-4) expression at mRNA and protein levels in the cells.Results There was a marked increase in IL-8 mRNA expression in HUVECs after 120 min of exposure to laminar flow shear stress. When exposed to shear stress for 180 min, there was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected endothelial cells. NF-κB p65 immunocytofluorescent staining of HUVECs showed that when exposed to the same flow shear stress for 30 or 60 min, the cell nuclei became stained; after 90 or 120 min, the staining became much more pronounced. A significant increase in P-IκB in the cell lysates occurred after 10 min of exposure while blot density dramatically dropped after 60 min of exposure. The density of the IκB blot dropped with increasing exposure time after 30 min. TLR-4 was present on the surface of HUVECs. HUVECs constitutively expressed TLR-2 and TLR-4 mRNA; when exposed to flow shear stress for 60 min, TLR-4 mRNA expression increased. Conclusion NF-κB activation is involved in flow shear stress-induced IL-8 mRNA expression in human umbilical vein endothelial cells. TLR-4 receptor for innate immunity most likely mediate these events.