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低氧激活巨噬细胞内NF-κB 信号转导通路的机制

张翠萍, 谢印芝, 陈鹏, 洪欣, 肖忠海, 马燕, 吕永达

军事医学科学院卫生学环境医学研究所,天津,300050

摘要

利用离体培养的巨噬细胞(macrophage)低氧培养模型(1%O2,5%CO2),采用2',7'-二氯荧光乙酰乙酸盐(2',7'-dichlorofluorescein diacetate,DCFH-DA)荧光分光光度法、Western blotting法和逆转录酶链反应(reversetranscription polymerasechain reaction,RT-PCR)法,观察低氧后细胞内活性氧(reactive oxygen species,ROS)水平、IκBα的酪氨酸(Tyr)磷酸化水平、P65 mRNA转录水平以及细胞核内NF-κB(nuclearfactor kappa B)激活量的变化,探讨低氧激活NF-κB信号转导通路的机制。

关键词： 低氧; 巨噬细胞; 活性氧; 核因子κB

Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation

Zhang Cuiping, Xie Yinzhi, Chen Peng, Hong Xin, Xiao Zhonghai, Ma Yan, Lu Yongda

Institute of Health and Environmental Medicine, Academy of Military Medical Sciences. Tianjin 300050, China；Institute of Radioactive Medicine, Academy of Military Medical Sciences. Beijing 100850, China

Abstract

The effects of hypoxia on the level of reactive oxygen species (ROS), IκBα tyrosine phosphorylation, transcription of P65 mRNA and NF-κB activation in isolated rat peritoneal macrophages were investigated by DCFH-DA fluorescence spectrophotometry, Western blotting and RT-PCR. The results obtained are as follows. (1) During hypoxia, the levels of intracellular ROS began to increase at 1 h, then reached a peak at 2 h, and began to decrease after 3 h. IκB#alpha# tyrosine phosphorylation began to rise after 2 h hypoxia and was the highest after 3 h hypoxia. After 4 h hypoxia it decreased gradually. NF-κB activation began to increase after 3 h hypoxia, and reached a peak after 4 h hypoxia. (2) When antioxidant NAC (500 #mu#mol/L) was added into the medium, the level of IκB#alpha# phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 #mu#mol/L), NF-κB activation induced by hypoxia was blocked significantly.

Key words: Hypoxia;Macrophages;Reactive oxygen species;Nuclear factor kappa B

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引用本文：

张翠萍, 谢印芝, 陈鹏, 洪欣, 肖忠海, 马燕, 吕永达. 低氧激活巨噬细胞内NF-κB 信号转导通路的机制[J]. 生理学报 2004; 56 (4): .

Zhang Cuiping, Xie Yinzhi, Chen Peng, Hong Xin, Xiao Zhonghai, Ma Yan, Lu Yongda. Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation. Acta Physiol Sin 2004; 56 (4): (in Chinese with English abstract).