胆固醇敏感器SCAP功能失调对THP-1源性巨噬细胞炎症因子表达的影响  

Dysfunction of sterol regulatory element-binding protein cleavage-activating protein promotes expression of inflammatory cytokines in THP-1 macrophages

作　　者：欧阳南[1] 杜晓刚[1] 陈利群[1] 周超[2] 

机构地区：[1]重庆医科大学附属第一医院肾脏内科,重庆400016 [2]重庆医科大学附属第一医院心血管内科,重庆400016

出　　处：《第三军医大学学报》2016年第5期449-455,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81500341)~~

摘　　要：目的观察胆固醇调节元件结合蛋白裂解激活蛋白(sterol regulatory element binding proteins cleavage-activating protein,SCAP)功能失调对THP-1源性巨噬细胞脂质代谢及IL-1β、MCP-1表达的影响,探讨SCAP的促炎作用是否依赖于其介导的胆固醇稳态调节功能。方法采用基因转染方法在THP-1源性巨噬细胞中过表达SCAP或沉默SCAP,结合使用炎症刺激剂LPS(1μg/m L)或SCAP转位抑制剂Betulin(3μmol/L)处理THP-1源性巨噬细胞,将细胞分为对照组、过表达SCAP组、沉默SCAP组、LPS组、沉默SCAP+LPS组、Betulin组以及过表达SCAP+Betulin组,RT-PCR法检测过表达SCAP或沉默SCAP对HMGCo AR、pro-IL-1β、MCP-1基因表达水平的影响。油红O染色观察不同组别细胞内中性脂质沉积的变化。酶法定量测定胞内胆固醇含量。Western blot法检测SCAP、pro-IL-1β以及培养上清中IL-1β的蛋白水平。结果 1过表达SCAP组与对照组比较,其HMGCo AR、MCP-1、proIL-1β基因表达水平升高(P<0.01),细胞内pro-IL-1β及细胞培养上清中IL-1β蛋白含量均上升(P<0.05),且细胞内总胆固醇(total cholesterol,TC)及胆固醇酯(cholesterol ester,CE)含量上升(P<0.01)。2沉默SCAP组与对照组比较,HMGCo AR、MCP-1、pro-IL-1β基因表达水平下降(P<0.05),细胞内proIL-1β及培养上清中成熟IL-1β蛋白含量均降低(P<0.05),且细胞内中性脂质蓄积减少,TC及CE含量下降(P<0.05);沉默SCAP+LPS组与LPS组比较,MCP-1、pro-IL-1β基因表达下调(P<0.05),成熟IL-1β蛋白水平下降(P<0.05)。过表达SCAP+Betulin组与过表达SCAP组比较,HMGCo AR基因表达水平下降(P<0.05),其细胞内,TC及CE含量亦下降(P<0.05),但MCP-1、pro-IL-1β基因表达水平差异无统计学意义(P>0.05),培养上清中成熟IL-1β蛋白含量差异也无统计学意义(P>0.05)。结论胆固醇敏感器SCAP功能失调促进THP-1源性巨噬细胞内炎症因子IL-1β、MCP-1表达,增加成熟IL-1β的生成及其向细胞外分泌;SCAP这一新发现的功能Objective To determine the effects of the dysfunction of sterol regulatory element-binding protein （SREBP） cleavage-activating protein （SCAP） on the expression of inflammatory cytokines, IL-1β and monocyte chemoattractant protein-1 （MCP-1）, in THP-1 macrophages, and investigate whether its pro-inflammatory effect depends on its mediated regulation of cholesterol homeostasis. Methods Gene transfection was used to over-express or knock-down SCAP in THP-1 macrophages. After THP-1 macrophages were treated with inflammatory stimulus, LPS （1 μg/mL） or translocation inhibitor, betulin （3 μmol/L, a chemical which inhibits SCAP translocation from endoplasmic reticulum to Golgi specifically）. The THP-1 macrophages were divided into control group, SCAP over-expression group, SCAP knocking-down group, SCAP knocking-down plus LPS group, and SCAP over-expression plus betulin group. The mRNA expression of SCAP, HMGCoAR, pro-IL-1β and MCP-1 were determined after over-expression or knocking-down of SCAP by real-time PCR. The accumulation of neutral lipid droplets in the cells was observed by Oil Red O staining. The contents of intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were determined by quantitative assays. The protein expression of SCAP and pro-IL-1β, and the mature IL-1β level in the supernatants were measured by Western blotting. Results Over-expression of SCAP significantly increased the expression of HMGCoAR, pro-IL-1β and MCP-1 at mRNA level （P〈0.01）, the contents of intracellular pro-IL-1β and mature IL-1β in the supernatants （P〈0.05）, and intracellular TC accumulation and CE contents in SCAP over-expressed macrophages. Knocking-down SCAP dramatically decreased the expression of HMGCoAR, pro-IL-1β and MCP-1 at mRNA level （P〈0.05）, the contents of intracellular pro-IL-1β and mature IL-1β in the supernatants （P〈0.05）, and intracellular TC accumulation and CE contents in SCAP knocking-down macrophages. The expressi

关 键 词：胆固醇调节元件结合蛋白裂解激活蛋白 白介素-1&beta 炎症 脂质代谢 巨噬细胞 

分 类 号：R341[医药卫生—基础医学] R392.11