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孤儿核受体Nur77通过转录激活功能调控巨噬细胞脂质沉积

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2014年第6期
页码:
625-630
栏目:
基础研究
出版日期:
2014-06-25

文章信息/Info

Title:
Lipid loading in macrophages is regulated by Nur77 nuclear import and its DNA binding activity
作者:
万 方; 胡刘华; 蔡招华; 聂 鹏; 沈玲红; 何 奔
(上海交通大学医学院附属仁济医院心内科,上海 200127)
Author(s):
WAN Fang;  HU Liu-hua;  CAI Zhao-hua;  NIE Peng;  SHEN Ling-hong;  HE Ben
(Department of Cardiology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China)
关键词:
Nur77; 巨噬细胞; 动脉粥样硬化; CD36; ABCA1
Keywords:
Nur77;  macrophage;  atherosclerosis;  CD36;  ABCA1
分类号:
R543.4
DOI:
-
文献标识码:
A
摘要:
目的:探讨孤儿核受体Nur77调控巨噬细胞脂质沉积与其转录激活功能的关系。方法: 建立稳定表达GFP、GFP-Nur77、GFP-Nur77/ΔDBD和GFP-Nur77/ΔTAD cDNA的小鼠巨噬细胞RAW264.7单克隆细胞系, 40 μg/ml氧化型低密度脂蛋白（ox-LDL）刺激24 h后，油红“O”染色定性观察细胞内脂质沉积，液相色谱-质谱联用法（LC-MS）定量检测细胞内胆固醇酯，荧光实时定量PCR (RT-PCR) 检测CD36和ABCA1 mRNA水平的变化，流式细胞仪检测CD36蛋白表达，Western blot方法检测ABCA1蛋白表达。 结果: 与对照GFP- RAW264.7相比，高表达Nur77能够显著减少ox-LDL诱导引起的巨噬细胞内脂质沉积，同时降低CD36的mRNA及蛋白表达，上调ABCA1的mRNA及蛋白表达。但GFP-Nur77/ΔDBD RAW264.7和GFP-Nur77/ΔTAD RAW264.7细胞内胆固醇都明显升高，并伴随CD36的mRNA及蛋白表达的降低和ABCA1的mRNA及蛋白表达的升高。结论: 孤儿核受体Nur77通过减少脂质摄取和增加脂质排出而减轻巨噬细胞内脂质沉积，这一作用与Nur77 DNA结合能力以及转录活性有关。
Abstract:
AIM:To investigate the role of Nur77 and its transcriptional function in lipid loading in macrophages. METHODS: We established four stable RAW264.7 strains expressing GFP, GFP-Nur77, GFP-Nur77/ΔDBD and GFP-Nur77/ΔTAD, respectively, by transfecting with corresponding plasmids. After 24 h stimulation by ox-LDL, intracellular lipid loading of each strain was observed by Oil red O dye. The intracellular cholesterol level was measured by liquid chromatographic-mass spectrometry (LC-MS). Transcriptional changes of CD36 and ABCA1 were monitored by real-time quantitative-PCR, and the expressions of these two proteins were assayed by flow cytometry and Western blot, respectively. RESULTS: Compared with GFP RAW264.7 cells, the high expression of Nur77 lowered the intracellular lipid loading in macrophages induced by ox-LDL, and downregulated CD36 and upregulated ABCA1 mRNA transcription and protein expression. However, the intracellular lipid loading in GFP-Nur77/ΔDBD and GFP-Nur77/ΔTAD RAW264.7 cells increased significantly with the increasing of CD36 mRNA and protein and the decreasing of ABCA1 mRNA and protein. CONCLUSION: Orphan nuclear receptor Nur77 reduces intracellular lipid loading in macrophages through decreasing cholesterol influx and increasing cholesterol efflux. The anti-atherosclerotic effect of Nur77 may be associated with its nuclear import and its DNA binding activity.

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备注/Memo

备注/Memo:
收稿日期：2014-02-28.
基金项目：国家自然科学基金项目资助（81070239，30971185） 通讯作者：何奔，主任医师，主要从事冠心病及介入治疗研究Email:heben@medmail.com.cn
作者简介：万方，住院医师，硕士Email:f5013039090@hotmail.com
更新日期/Last Update: 2014-07-10