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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。
Objective To investigate the effect of angiotensin Ⅱ on transcription factor NF κB and the expression of platelet-derived growth factor B chain (PDGF B) gene in ECV3 04 cells. Methods Electrophoretic mobility shift assay (EMSA), immunohistochemistry, confocal microscopy and gold particle-labeled immunoelectron microscopy were used. The co-transfection of luciferase reporter gene and mutant kinase plasmid and Northern blotting were used to study the effects of vascular tension The expression of NF-κB in ECV3 0 4 cells was detected by radioimmunoassay and the expression of PDGF B mRNA in ECV3 0 4 cells before and after stimulation with angiotensin Ⅱ was detected. Results The activation of NF-κB and nuclear translocation in ECV3 0 4 cells were stimulated by angiotensin Ⅱ. 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 inhibited luciferase expression in NF-κB-activated transfected cells stimulated by angiotensin Ⅱ. Conclusion Angiotensin Ⅱ activates NF κB in the cytoplasm and translocates to nuclear translocation. Kinase NIK, IKKα and IKKβ participate in this signaling pathway. Angiotensin Ⅱ stimulated PDGF B chain mRNA levels increased.