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目的和方法 :分别采用凝胶迁移率改变法 (EMSA)和Western -blot的方法从基因转录水平探讨AngII对血管内皮细胞株ECV30 4的影响。结果 :(1)EMSA结果表明 ,AngII组核抽提物与 3种探针的结合活性均高于正常对照组 ,与NF -κB ,SP - 1和AP - 1探针结合活性分别为正常对照组的 10 98倍 ,3 97倍和 1 33倍 ,说明AngII对血管内皮细胞的作用机制可能与NF -κB ,SP - 1和AP - 1等转录因子的转录活性增加有关。进一步证实NF -κB ,AP -1在内皮细胞功能失调发病机制中的重要作用 ,同时发现转录因子SP - 1也可能参与此过程。 (2 )Western -blot结果表明 ,AngII处理后细胞核内NF -κB含量明显增加 ,提示AngII能够增加血管内皮细胞转录因子NF -κB的核转位 ,进一步验证了EMSA的结论。而细胞核内SP - 1和AP - 1的含量较对照组略有增加 ,提示AngII对SP - 1和AP - 1的核转位无明显影响而可能主要是促进转录因子与相应顺式调控元件的结合活性。结论 :AngII对内皮细胞 3种重要的转录因子的活性有不同程度的影响 ,这在心血管疾病的发病中可能起着重要的作用
Purpose and Methods: The effect of AngII on endothelial cell line ECV30 4 was investigated by gene-transcriptional level using EMSA and Western-blot respectively. Results: (1) The result of EMSA showed that the binding activity of nuclear extract of AngII group with three probes was higher than that of normal control group. The binding activity to NF - κB, SP - 1 and AP - 1 probes were normal control The results showed that the mechanism of AngII on vascular endothelial cells may be related to the increase of transcriptional activity of NF - κB, SP - 1 and AP - 1. It further confirmed the important role of NF-κB and AP-1 in the pathogenesis of endothelial dysfunction and found that the transcription factor SP-1 may also participate in this process. (2) The result of Western-blot showed that the content of NF-κB in the nucleus increased remarkably after AngII treatment, suggesting that AngII could increase the nuclear translocation of NF-κB, which further confirmed the conclusion of EMSA. However, the content of SP - 1 and AP - 1 in the nucleus increased slightly compared with the control group, suggesting that AngII had no obvious effect on the nuclear translocation of SP - 1 and AP - 1 but probably mainly promoted the transcription factor and cis - regulatory elements Binding activity. CONCLUSION: AngII has different effects on the activity of three important transcription factors of endothelial cells, which may play an important role in the pathogenesis of cardiovascular diseases