不同剂量TNF-α对Nrf2转录调节活性的影响

Regulation of transcriptional factor NF-E2-related factor 2 by different doses of TNF-α

目的:研究不同剂量肿瘤坏死因子-α(TNF-α)对肺微血管内皮细胞NF-E2相关因子2(Nrf2)转录调节活性的影响。方法:肺组织块贴壁法培养大鼠肺微血管内皮细胞,Ⅷ因子相关抗原间接免疫荧光鉴定所培养的细胞。采用含有0μg/L、2.5μg/L、5μg/L、10μg/L、20μg/L和40μg/LTNF-α的无血清培养基处理肺微血管内皮细胞4h后,以免疫细胞化学技术观察细胞内Nrf2的亚定位及含量的变化;提取细胞核蛋白和总RNA,以变动迁移率分析技术检测Nrf2转录调节活性的变化以及采用RT-PCR技术检测细胞Nrf2 mRNA表达水平的变化。结果:采用2.5、5.0、10.0μg/LTNF-α处理细胞,核内Nrf2水平和Nrf2转录调节活性随浓度的增加而呈现上升趋势;而采用20.0μg/L、40.0μg/LTNF-α处理细胞,核内Nrf2水平及其转录调节活性有所下降;不同剂量TNF-α对细胞Nrf2基因转录水平没有明显影响。结论:不同剂量TNF-α对肺微血管内皮细胞Nrf2转录调节活性产生不同影响:中、低剂量TNF-α增强Nrf2转录调节活性,而大剂量TNF-α引起Nrf2转录调节活性下降。

AIM：To study the effects of tumor necrosis factor-α （TNF-α） on the transcriptional activity of nuclear factor-erythroid 2-related factor 2 （Nrf2） in pulmonary microvascular endothelial cells. METHODS： Rat pulmonary micro-vascular endothelial cells （PMVECs） were cultured by lung tissue block pasted methods,and identified immunocytochemically using Ⅷ factor-related antigen. The cells were treated with different doses TNF-α （prepared in serum-free medium） for 4 h. Subcellular localization and levels of Nrf2 in PMVECs were observed with immunocytochemical methods. Nuclear extract were obtained to assayed transcriptional activity of Nrf2 with EMSA. Total RNA were isolated to assay the mRNA expression of Nrf2 by RT-PCR. RESULTS： The protein level of Nrf2 in the nuclei and transcriptional activity increased dose-dependently in PMVECs after treated with TNF-α at concentrations of 2.5,5.0 or 10.0 μg/L. However,the protein level of Nrf2 in nuclei and transcriptional activity decreased dose-dependently in PMVECs after treated with TNF-α at concentrations of 20 or 40 μg/L. No different mRNA expression of Nrf2 in PMVECs treated with TNF-α at all concentration above was observed. CONCLUSION： Transcriptional activity of Nrf2 increases in PMVECs treated with low or moderate doses of TNF-α and decreases in PMVECs treated with high doses of TNF-α.