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目的本研究检测层流低切应力诱导人脐静脉血管内皮细胞IL8基因的转录激活。方法RTPCR检测4.2dyne/cm2切应力处理0.5、1、2h人脐静脉血管内皮细胞的IL8mRNA表达。构建IL8报告基因质粒pEGFP1IL8USCS,转染脐静脉血管内皮细胞,切应力刺激3h后,流式细胞仪分析绿色荧光蛋白表达。免疫荧光细胞化学染色观察切应力处理脐静脉血管内皮细胞0.5、1、1.5、2h的NFκBp65核转移。用对照和切应力处理10、20、30、60min的脐静脉血管内皮细胞胞质蛋白,进行IκB和磷酸化IκB的免疫印迹。结果低切应力刺激可诱导脐静脉血管内皮细胞表达IL8mRNA。切应力刺激后,重组质粒转染的血管内皮细胞表达IL8绿色荧光蛋白报告基因。切应力诱导NFκB向胞核内转移,和IκB的磷酸化和降解。结论NFκB传导通路可能介导切应力诱导脐静脉血管内皮细胞IL8基因的转录活化,参与动脉粥样硬化的形成。
Objective This study was designed to investigate the transcriptional activation of IL8 gene in human umbilical vein endothelial cells induced by laminar low shear stress. Methods RTPCR was used to detect the IL8 mRNA expression in human umbilical vein endothelial cells at 0.5 dyne / cm2 shear stress. IL8 reporter gene plasmid pEGFP1IL8USCS was constructed and transfected into umbilical vein endothelial cells. After stimulated by stress for 3h, the expression of green fluorescent protein (GFP) was analyzed by flow cytometry. Immunofluorescence cytochemical staining was used to observe the nuclear translocation of NFκBp65 at 0.5, 1, 1.5, 2 h after stress-induced shear stress in umbilical vein endothelial cells. Immunofluorescence of IκB and phosphorylated IκB was performed with control and shear stress for 10, 20, 30, 60 min of umbilical vein endothelial cell cytoplasmic proteins. Results Low shear stress induced the expression of IL8 mRNA in umbilical vein endothelial cells. After the shear stress stimulation, recombinant plasmid transfected vascular endothelial cells express the IL8 green fluorescent protein reporter gene. Shear stress induces the transfer of NFKB to the nucleus, and the phosphorylation and degradation of IKB. Conclusion The NFκB pathway may mediate the transcriptional activation of IL8 gene in human umbilical vein endothelial cells induced by shear stress, which is involved in the formation of atherosclerosis.