摘要
背景抑制巨噬细胞脂质蓄积(泡沫化)和炎症因子释放是防治动脉粥样硬化(AS)的重要途径。微小RNA(miR)-146a是否通过三磷酸腺苷结合盒转运体A1(ABCA1)等膜蛋白以三磷酸腺苷(ATP)为能源将细胞内游离胆固醇转运到细胞膜表面,进而减少细胞内胆固醇蓄积尚未知。目的探讨微小RNA(miR)-146a是否通过靶向沉默ABCA1调控THP-1巨噬细胞源性泡沫细胞胆固醇流出。方法本次实验时间为2018年12月—2020年6月。培养THP-1细胞(人单核细胞株)并经佛波酯(PMA)诱导分化为巨噬细胞,其吞噬脂质形成巨噬细胞源性泡沫细胞。将巨噬细胞源性泡沫细胞分为空白对照组,培养液中不加任何其他试剂;阳性对照组,培养液中加入肝X受体激动剂T0901317;阴性对照组,培养液中加入ABCA1小干扰RNA(siRNA);模拟物组,培养液中加入miR-146a mimics;抑制剂组,培养液中加入miR-146a inhibits;采用液体闪烁计数法检测胆固醇流出效率。将巨噬细胞源性泡沫细胞分别接种到6孔板并培养至生长对数期,按照要求分别加入In-NC(NC组)、miR-146a mimics(模拟物组)、miR-146a inhibits(抑制剂组)、miR-146a mimics+核因子κB(NF-κB)抑制剂(模拟物+PDTC组)、miR-146 anhibits+NF-κB抑制剂(抑制剂+PDTC组),分别采用实时荧光定量聚合酶链式反应(RT-PCR)和Western blotting法检测NF-κB通路p50、p65及ABCA1的mRNA、蛋白相对表达量。将巨噬细胞源性泡沫细胞接种于培养瓶内,汇合度达到80%后分别转染miR-146a mimics(模拟物组)、miR-146a inhibits(抑制剂组)及In-NC(NC组),采用Annexin V-FITC/PI双染流式细胞术检测正常细胞、坏死细胞及凋亡细胞水平。采用荧光素酶报告基因检测miR-146a与ABCA1结合情况。结果阳性对照组胆固醇流出效率高于空白对照组,阴性对照组和模拟物组胆固醇流出效率低于阳性对照组,抑制剂组胆固醇流出效率高于空白对照组、阴性对照组和模拟物组(P<0.05)。模拟�
Background Inhibiting lipid accumulation(foaming)of macrophages and the release of inflammatory factors are important way to prevent and treat atherosclerosis(AS).Whether MicroRNA(miR)-146a uses ATP as an energy source to transport free cholesterol in cells to the surface of cell membranes through membrane proteins such as ABCA1,which can significantly reduce the accumulation of cholesterol in cells is still unknown.Objective To explore whether miR-146a targets ABCA1 and regulates intracellular cholesterol efflux in THP-1 macrophage-derived foam cells.Methods The experiment time is from December 2018 to June 2020.Culture THP-1 cells(human monocyte cell line)and differentiate into macrophages induced by phorbol ester(PMA),which engulf lipids to form macrophage-derived foam cells.Divide the macrophage-derived foam cells into a blank control group,without any other reagents in the culture medium;positive control group,add liver X receptor agonist T0901317 to the culture medium;negative control group,add ABCA1 small interference to the culture medium RNA(siRNA);mimic group,miR-146a mimics was added to the culture solution;inhibitor group,miR-146a inhibits was added to the culture solution;liquid scintillation counting method was used to detect the efficiency of cholesterol efflux.Inoculate macrophage-derived foam cells into six-well plates and culture them to the logarithmic growth phase.Add In-NC(NC group),miR-146a mimics(mimic group),miR-146a inhibits(inhibitior group),miR-146a mimics+NF-κB inhibitor(mimic+PDTC group),miR-146 anhibits+NF-κB inhibitor(inhibitor+PDTC group),real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and Western blotting were used to detect the relative expression of mRNA and protein of p50 and p65 in the NF-κB pathway.The macrophage-derived foam cells were inoculated into the culture flask,and after the confluence reached 80%,they were transfected into miR-146a mimics(mimic group),miR-146a inhibits(inhibitor group)and In-NC(NC group).Annexin V-FITC/PI double staining flow
作者
幸世峰
孙理华
骆小梅
XING Shifeng;SUN Lihua;LUO Xiaomei(Department of Cardiovascular,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830000,China)
出处
《实用心脑肺血管病杂志》
2021年第5期63-69,共7页
Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease
基金
新疆维吾尔自治区自然科学基金面上项目(2018D01C308)。
