M2型巨噬细胞来源的外泌体对快速起搏HL-1细胞KCa3.1的作用及可能机制  

M2 macrophage-derived exosomes regulate the KCa3.1 channel in the rapid pacing HL-1 myocytes

作　　者：陈慧宇 刘弟世闻 姚亚君 付韫韬 何山青 赵庆彦[1] CHEN Hui-yu;LIU Di-shi-wen;YAO Ya-jun;FU Yun-tao;HE Shan-qing;ZHAO Qing-yan(Department of Cardiology,Renmin Hospital of Wuhan University,Cardiovascular Research Institute,Wuhan University,Hubei Key Laboratory of Cardiology,Wuhan 430060,Hubei,China)

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

出　　处：《中国心脏起搏与心电生理杂志》2023年第3期224-231,共8页Chinese Journal of Cardiac Pacing and Electrophysiology

基　　金：国家自然科学基金(82170312)。

摘　　要：目的探究M2型巨噬细胞来源的外泌体对快速起搏小鼠心房肌细胞(HL-1)KCa3.1的作用及可能机制.方法提取C57/6J小鼠骨髓巨噬细胞并分为未分化组(未加干预的骨髓巨噬细胞)和分化组(骨髓巨噬细胞经白细胞介素-4干预24h分化为M2型巨噬细胞).分别收集分化组和未分化组细胞培养液上清,采用超速离心法得到外泌体.实时荧光聚合酶链式反应(qRT-PCR)检测miR-146a-5p在分化组和未分化组外泌体中的表达情况.将HL-1细胞分为8组,分别为对照组(HL-1细胞未施加任何干预)、起搏组(HL-1细胞快速起搏)、共培养组(HL-1细胞快速起搏的同时与M2型巨噬细胞来源的外泌体共培养)、模拟物组(HL-1细胞快速起搏同时转染miR-146a-5p模拟物)、模拟物对照组(HL-1细胞快速起搏同时转染模拟物对照)、抑制物组(HL-1细胞快速起搏同时转染miR-146a-5p抑制物)、抑制剂物对照组(HL-1细胞快速起搏同时转染抑制剂物对照)、PDTC组[HL-1细胞起搏同时给予核因子-κBp65(NF-κB p65)特异性阻断剂PDTC].采用qRT-PCR检测各组中miR-146a-5p相对表达量.免疫荧光检测共培养组中HL-1细胞对外泌体的摄取情况.蛋白质免疫印迹检测上述各组KCa3.1和磷酸化核因子-κBp65(p-NF-κBp65)的表达情况.全细胞膜片钳记录各组KCa3.1的电流密度.结果与未分化组比较,分化组外泌体miR-146a-5p表达量更高(P<0.01).与对照组比较,起搏组KCa3.1和p-NF-κBp65蛋白相对表达水平和KCa3.1的电流密度明显升高(P<0.001).与起搏组比较,共培养组和PDTC组KCa3.1和p-NF-κBp65的蛋白相对表达水平和KCa3.1的电流密度明显降低(P均<0.05).与模拟物对照组比较,模拟物组KCa3.1和p-NF-κBp65的蛋白相对表达水平和KCa3.1的电流密度明显下调(P均<0.05).与抑制物对照组比较,抑制物组明显提高了KCa3.1和p-NF-κBp65蛋白相对表达水平和KCa3.1的电流密度(P均<0.01).结论M2型巨噬细胞外泌体及其所携带的miR-146a-Objective To investigate the role and mechanism of M2 macro phage-derived exosomes on KCa3.1channel in the pacing mouse atrial myocytes(HL-1).Methods Bone marrow macrophages from the tibia and femur of C57/6J mice were isolated and divided into the undifferentiated group(untreated bone marrow macrophages)and differentiated group(bone marrow macrophages were differentiated into M2 macrophages with interleukin-4 for24 h).The cell culture medium supernatant of the differentiated group and the undifferentiated group was collected,and the exosomes were obtained by ultracentrifugation.Real-time fluorescent polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-146a-5p in the exosomes in the differentiated group and the undifferentiated group.HL-1 cells were divided into 8 groups:control group(without any treatment),pacing group(HL-1 cells were rapidly paced),co-culture group(HL-1 cells were rapidly paced and co-cultured with exosomes derived from M2 macrophages),mimic group(HL-1 cells were paced and transfected with miR-146a-5p mimics),and mimic control group(HL-1 cells were paced and transferred simultaneously),inhibitor group(HL-1 cells were paced and transfected with miR-146a-5p inhibitor),inhibitor control group(HL-1 cells were paced and transfected with inhibitor control),and PDTC group(HL-1 cells were paced and treated with nuclear factor-κb(NF-κB p65)specific blocker,PDTC).qRT-PCR was used to detect the relative expression of miR-146a-5p in each group.The expression of KCa3.1 and p-NF-κB p65 in each group was detected by Western blot.Whole-cell patch clamp was used to record the current density of KCa3.1 in each group.Results Compared with the control group,the protein expression levels of KCa3.1 and p-NF-κBp65 and the current density of KCa3.1 were significantly increased in the pacing group(P<0.001).Compared with the undifferentiated group,the differentiated group had a significantly higher expression of exosomal miR-146a-5p(P<0.01).The protein expression of KCa3.1 and p-NF-κB p65 and

关 键 词：心血管病学 M2型巨噬细胞外泌体 KCa3.1 电重构 心房颤动 

分 类 号：R541.75[医药卫生—心血管疾病] R331.38[医药卫生—内科学]