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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 2dyne/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 8mRNA表达情况。同时构建IL 8上游调控序列 (IL 8USCS)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 12 0min后 ,内皮细胞表达IL 8mRNA明显增强。切应力刺激 180min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 30min ,胞核即出现阳性反应 ,刺激 90min后 ,胞核呈强阳性染色。切应力刺激 10min时磷酸化IκB即显著增强 ,60min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 60min后 ,TLR 4mRNA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T
Objective To detect the gene expression of interleukin 8 (IL 8) induced by shear stress in human umbilical vein endothelial cells (VECs) and to explore the mechanism of its transcriptional activation. Methods 4 2dyne / cm2 laminar shear stress was used to stimulate cells. RT-PCR was used to detect the expression of IL 8mRNA in endothelial cells after shear stress. At the same time, we constructed the green fluorescent enhanced protein (EGFP) reporter gene pEGFP1 IL8USCS of IL 8USCS. The transcriptional activation of IL 8 gene was detected by transgene and flow cytometry. The nuclear translocation of NF-κB was observed by immunofluorescence cytochemical staining. Western blotting detects shear stress-induced phosphorylation and degradation of IKB. Toll-like receptor 4 (TLR4) expression was detected by RT PCR, Northern blot and immunofluorescence cytochemistry at the mRNA and protein levels. Results After stimulated with shear stress for 120 minutes, the expression of IL 8 mRNA in endothelial cells was significantly increased. The green fluorescent protein expression of vascular endothelial cells transfected with pEGFP1 IL8USCS was significantly enhanced after 180min stimulation with shear stress. NF-κB 5 immunofluorescence cytochemical staining showed that the nucleus appeared a positive reaction after 30 min stimulation with shear stress, and the nuclei showed strong positive staining after 90 min stimulation. The IκB phosphorylation was significantly enhanced at 10 min after shear stress, and the intensity of IκB phosphorylation disappeared after 60 min, while the IκB decreased gradually with the prolongation of stimulation time. Toll-like receptor 2 (TLR 2) and TLR 4 receptor were expressed on the surface of umbilical vein endothelial cells. The expression of TLR 4 mRNA was significantly enhanced after 60 min stimulation with shear stress. Conclusion Fluid shear stress stimulates the expression of IL 8 gene in vascular endothelial cells, which is related to the activation of NF κB. The natural immune receptor T