M1型巨噬细胞来源的外泌体微小RNA-16-5p对心房肌细胞电生理的影响  

Effects of M1 macrophage-derived exosome microRNA-16-5p on electrophysiology of atrial myocytes

作　　者：程燕妮 王学文 曹真 付韫韬 柯元甲 郭珂欣 李雅佳 龙晓建 赵庆彦[1] Cheng Yanni;Wang Xuewen;Cao Zhen;Fu Yuntao;Ke Yuanjia;Guo Kexin;Li Yajia;Long Xiaojian;Zhao Qingyan(Department of Cardiology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院心血管内科、武汉大学心血管病研究所、心血管病湖北省重点实验室,武汉430060

出　　处：《临床内科杂志》2024年第1期51-55,共5页Journal of Clinical Internal Medicine

基　　金：国家自然科学基金资助项目(82170312)。

摘　　要：目的 探讨M1型巨噬细胞来源的外泌体微小RNA(miR)-16-5p对心房肌细胞电生理的影响及可能机制。方法 将小鼠单核巨噬细胞(RAW264.7细胞)分为脂多糖(LPS)组(LPS诱导刺激RAW264.7细胞24 h使其分化为M1型巨噬细胞)、miR-16-5p阴性对照(NC)组(将miR-16-5p NC转染RAW264.7细胞后诱导分化)、miR-16-5p模拟物(mimics)组(将miR-16-5p mimics转染RAW264.7细胞后诱导分化)、miR-16-5p抑制物(inhibitor)组(将miR-16-5p inhibitor转染RAW264.7细胞后诱导分化)及LPS+中性鞘磷脂酶抑制剂(GW4869)组(RAW264.7细胞经LPS诱导完成后加入10μM GW4869继续培养)。5组巨噬细胞培养48~72 h后收集上清液,采用超速离心法提取外泌体。采用实时荧光PCR(qRT-PCR)检测转染后巨噬细胞miR-16-5p相对表达水平。将心房肌细胞(HL-1细胞)暴露于快速电刺激(1.0 V/cm, 10 Hz)48 h构建快速起搏诱导的房颤细胞模型,与各组外泌体共培养,心房肌细胞分为A组(起搏HL-1细胞+LPS组外泌体)、B组(起搏HL-1细胞+miR-16-5p NC组外泌体)、C组(起搏HL-1细胞+miR-16-5p mimics组外泌体)、D组(起搏HL-1细胞+miR-16-5p inhibitor组外泌体)、E组(起搏HL-1细胞+LPS+GW4869组外泌体)。采用膜片钳检测各组心房肌细胞动作电位时限[复极化达50%和90%的动作电位时限(APD50、APD90)],采用蛋白质免疫印记法检测5组细胞中磷脂酰肌醇激酶(PI3K)、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)表达水平。结果 miR-16-5p mimics组巨噬细胞miR-16-5p相对表达水平高于miR-16-5p NC组(P<0.001),而miR-16-5p inhibitor组低于miR-16-5p NC组(P<0.05)。A组和B组HL-1细胞APD50和APD90比较差异均无统计学意义(P>0.05);C组HL-1细胞APD50和APD90均短于A组(P<0.05);D组及E组HL-1细胞APD50和APD90均长于A组(P<0.01)。A组和B组HL-1细胞PI3K表达水平、p-AKT/AKT比较差异均无统计学意义(P>0.05);C组HL-1细胞PI3K表达水平、p-AKT/AKT均低于A组(P<0.001);D组及E组HL-1细胞PI3K表达水平、p-AKT/AKT均高于A组(P<0Objective To explore the effects of M1 macrophage-derived exosome microRNA(miR)-16-5p on electrophysiology of atrial myocytes and explore the potential mechanisms.MethodsMononuclear macrophages(RAW264.7 cells) were divided into lipopolysaccharide(LPS) group(RAW264.7 cells were induced and stimulated by LPS for 24 h to differentiate into M1 macrophages),miR-16-5p negative control(NC) group(miR-16-5p NC was transfected into RAW264.7 cells and induced differentiation),miR-16-5p mimics group(miR-16-5p mimics were transfected into RAW264.7 cells and induced differentiation),miR-16-5p inhibitor group(miR-16-5p inhibitor was transfected into RAW264.7 cells and induced differentiation) and LPS+neutral sphingomyelin inhibitor(GW4869) group(RAW264.7 cells were induced by LPS and then cultured with 10 μM GW4869).Supernatant was collected after culture of macrophages for 48-72 h in 5 groups, and exosomes were obtained by ultracentrifugation.Real-time fluorescent PCR(qRT-PCR) was used to detect relative expression level of miR-16-5p in transfected macrophages.Atrial myocytes(HL-1 cells) were exposed to rapid electrical stimulation(1.0 V/cm, 10 Hz) for 48 h to construct rapid pacing-induced atrial fibrillation cell model, then co-cultured with exosomes from each group.Atrial myocytes were divided into A group(pacing HL-1 cells+exosomes from LPS group),B group(pacing HL-1 cells+exosomes from miR-16-5p NC group),C group(pacing HL-1 cells+exosomes from miR-16-5p mimics group),D group(pacing HL-1 cells+exosomes from miR-16-5p inhibitor group) and E group(pacing HL-1 cells+exosomes from LPS+GW4869 group).Patch clamp was used to detect action potential duration at 50% and 90% repolarization(APD_(50) and APD_(90)).Western blotting was uesd to detect expression levels of phosphatidylinositol kinase(PI3K),protein kinase B(AKT),phosphorylated AKT(p-AKT) in cells of 5 groups.Results Relative expression level of miR-16-5p in macrophages of miR-16-5p mimics group was higher than that of miR-16-5p NC group(P<0.001),while miR-16-5p inhibito

关 键 词：心房颤动 巨噬细胞 外泌体 动作电位 电重构 

分 类 号：R541.7[医药卫生—心血管疾病]