【摘要】 目的　研究血管紧张素Ⅱ对ECV3 0 4细胞中转录因子NF κB的作用和对血小板源生长因子B链 (PDGF B)基因表达的影响。方法　采用电泳迁移率移动分析法 (EMSA) ,免疫组织化学方法 ,共聚焦显微镜及金颗粒标记免疫电镜技术 ;荧光素酶报告基因与变异型激酶质粒共转染的方法及Northern印迹法研究血管紧张素Ⅱ激活ECV3 0 4细胞NF κB的信号传递路径和检测了血管紧张素Ⅱ刺激前后ECV3 0 4细胞PDGF BmRNA的表达水平。结果　血管紧张素Ⅱ刺激后 ,在ECV3 0 4细胞内有NF κB的激活及核易位过程 ,应用免疫荧光共聚焦显微镜 ,免疫电镜及Northern印迹等方法均可观察到PDGF B或其基因表达增高。变异型激酶质粒IKKα KM ,IKKβ KM及NIK Km可抑制经血管紧张素Ⅱ刺激的转染细胞内与NF κB启动相连的荧光素酶的表达。结论　血管紧张素Ⅱ可激活胞质内NF κB并出现核易位 ,激酶NIK、IKKα 和IKKβ 参与了此信号传递路径。血管紧张素Ⅱ刺激后PDGF B链mRNA水平增高。更多还原

【Abstract】 Objectives The renin angiotensin system may contribute to the pathogenesis of atherosclerosis. The transcription factor nuclear factor kappa B (NF κB) participates in most signal pathways involved in the inflammatory process. In this project the effect of angiotensin Ⅱ(Ang Ⅱ) on NF κB activation and the promotion of PDGF B mRNA expression in human endothelial cell line ECV304 was studied. Methods Electrophoretic mobility shift assay, immunofluorescence and immunoelectronic microscope techniques, including confocal microscopy and gold particle labelled electronic microscopy were applied to investigate the mechanism by which Ang Ⅱactivates NF κB, ECV304 cells were transiently transfected with an NF κB/luciferase reporter gene and catalytically inactive NIK, IKK α, IKK β mutants respectively. Northern blot was carried out to detect PDGF B mRNA. Results By the findings of immunofluorescence confocal microscopy, immunoelectronic microscopy and Northern blot, Ang Ⅱwas effective in stimulating NF κB activation and there was definited cytoplasmic to nuclear translocation of NF κB subunits p50 and p65 and overexpression of PDGF B mRNA expression. Over expression of the transiently transfected IKK α KM, IKK β KM and NIK KM mutant genes enabled to block the reporter gene activation induced by ang Ⅱ. Conclusion Ang Ⅱ is effective to activate NF κB through a pathway dependent on NIK, IKK α and IKK β, and induces PDGF B transcription in the endothelial cell line ECV304.更多还原