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目的 研究血管紧张素Ⅱ对ECV30 4细胞中转录因子NF κB的作用和对PDGF B基因表达的影响。方法 采用电泳迁移率移动分析法 (EMSA)和免疫组化方法 ,包括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NF κB的信号传递路径和检测了血管紧张素Ⅱ刺激前后PDGF BmRNA的表达水平。结果 血管紧张素Ⅱ刺激后 ,在ECV30 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AngⅡ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 AngⅡ可激活胞浆内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。
Objective To investigate the effect of angiotensin Ⅱ on transcription factor NFκB in ECV304 cells and its effect on PDGF B gene expression. Methods Electrophoretic mobility shift assay (EMSA) and immunohistochemical methods including confocal microscopy and gold particle-labeled immunoelectron microscopy, co-transfection of luciferase reporter gene and mutant kinase plasmid and Northern blot Tensin II activated the NF κB signaling pathway and detected the expression of PDGF B mRNA before and after angiotensin II stimulation. Results After stimulation with angiotensin Ⅱ, NFκB activation and nuclear translocation occurred in ECV304 cells. The expression of PDGF B or its gene was observed by immunofluorescence confocal microscopy, immunoelectron microscopy and Northern blotting. Mutant kinase plasmids IKKα KM, IKKβ KM and NIK KM could inhibit the expression of luciferase linked with NF κB activation in AngⅡ-stimulated transfected cells. Conclusions Ang Ⅱ activates NF κB in cytoplasm and translocates to nuclear translocation. The kinase NIK, IKKα and IKKβ participate in this signal pathway. Angiotensin Ⅱ stimulated PDGF B chain mRNA levels increased.