NRF2基因启动子-617C／A基因多态性对内毒素介导巨噬细胞炎症反应的影响  

Effects of NF-E2-related factor-2 promoter polymorphism on lipopolysaccharide-induced inflammatory responses in macrophages

作　　者：邱俏檬[1] 郑金韬[1] 南超[1] 赵媛媛[1] 赵光举[1] 洪广亮[1] 梁欢[1] 李萌芳[1] 卢中秋[1] 

机构地区：[1]温州医学院附属第一医院急诊医学中心,浙江省温州市325000

出　　处：《中华医学杂志》2013年第14期1114-1117,共4页National Medical Journal of China

基　　金：浙江省自然科学基金（Y2090927）;浙江省科技厅公益性技术应用研究计划（2010C33014）;温州市科技计划（Y20080088）;温州市高层次人才创新技术重点项目

摘　　要：目的 探讨人核因子E2相关因子（NRF2）基因启动子-617C／A多态性的功能，并观察其对内毒素诱导的巨噬细胞炎症反应的影响。方法构建分别携带NRF2基因启动子-617C、-617A诱导调控系统的质粒及腺病毒载体。应用构建好的质粒转染HEK293细胞后，以双荧光素酶分别验证NRF2基因启动子-617C／A多态的调控效率；应用构建好的腺病毒载体转染RAW264．7细胞，反转录-聚合酶链反应（RT—PCR）和Western印迹检测NRF2mRNA和蛋白表达水平；内毒素刺激巨噬细胞后，应用Western印迹检测NRF2蛋白的表达，ELlSA法检测细胞培养上清液中肿瘤坏死因子（TNF）-d、白细胞介素（IL）-6和IL-10的水平。结果NRF2基因启动子617C组的调控效率显著高于-617A组（0．584±0．016与0．258±0．018，P〈0．05）。内毒素刺激RAW264．7细胞后，-617C组NRF2蛋白和基因水平表达均明显高于-617A组（1．123±0．080与0．951±0．057，1．889±0．031与1．647±0．323，均P〈0．05）。-617C组上清IL-6、IL-10、TNF-0I水平均低于-617A组，其中IL-6、IL-10的表达差异均有统计学意义（均P〈0．05）。-617C组IL-6／IL-10也显著低于-617A组（P〈0．05）。结论NRF2基因启动子-617C／A多态性对NRF2的表达具有显著影响，并对内毒素刺激后巨噬细胞炎症反应的平衡有着重要调控作用。Objective To explore the effects of NF-E2-related factor-2 （NRF2） - 617C/A promoter polymorphism on NRF2 expression as well as lipopolysaccharide-induced inflammatory responses in macrophages. Methods NRF2 -617C/A promoter fragments were synthesized by chemical method and cloned into a pUC57 vector. The dul-luciferase reporter assay was employed to determine the activity of promoters. Then recombinant adenoviral vectors were constructed and transfected into macrophages. The expression of Nrf2 was examined by Western blotting and reverse transcription （RT） -PCR. The expressions of tumor necrosis factor-alpha （TNF-ct） , interleukin-6 （IL-6） and interleukin-10 （IL-10） in macrophages after the stimulation of LPS were determined by enzyme-linked immunosorbent assay （ELISA）. Results The activity of NRF2 -617C promoter-luciferase reporter （ FLuc/RLuc activity ratio ） was significantly higher than that of NRF2 - 617A group （0. 584 ± 0. 016 vs 0. 258 ± 0. 018, P 〈 0.05 ）. The NRF2 protein and mRNA levels in - 617C group were much higher than those of 617A group （ 1. 123 ＋ 0. 080 vs 0. 951 ＋ 0. 057,1. 889±0. 031 vs 1. 647 ±0. 323, both P 〈0. 05）. After the stimulation of LPS, the NRF2 protein expression in macrophages significantly increased （0. 584 ± 0.016 vs 0. 258 ± 0. 018, P 〈 0. 05 ）. Compared with -617A group, there was a significantly higher expression of NRF2 in -617C group （0. 671 ±0. 033 vs 0. 751 ± 0. 014, P 〈 0. 05 ）. Additionally, the productions of IL-6 and IL-10 in - 617C group were markedly lower than those in -617A group as well as IL-6/IL-10 （both P 〈 0. 05）. However, no significant difference existed in the levels of TNF-a between - 617C and - 617A groups （ P 〉 0. 05 ）. Conclusions The -617C/A promoter polymorphism of NRF2 may influence the NRF2 expression. And it appears to be associated with the LPS-induced inflammatory responses in macrophages.

关 键 词：启动子(遗传学) 多态性 单核苷酸 炎症 

分 类 号：R363[医药卫生—病理学]