microRNA-125b调控心肌梗死后炎症的作用及其在不同心脏组成细胞中的调控作用

发布时间：2017-12-28 09:38

本文关键词：microRNA-125b调控心肌梗死后炎症的作用及其在不同心脏组成细胞中的调控作用　出处：《中国细胞生物学学报》2016年12期 　论文类型：期刊论文

【摘要】：该研究旨在探究小鼠微小RNA-125b(microRNA-125b,miR-125b)调控心肌梗死后,内源性危险因子即高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)释放诱导的炎症反应及其在3种心脏主要组成细胞中调控作用的比较。首先建立小鼠心梗模型,检测心梗早期miR-125b及炎性细胞因子白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-12(interleukin-12,IL-12)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-a)的表达变化并观察心梗后miR-125b过表达对心功能的影响;用免疫组织化学和免疫荧光方法检测小鼠心肌梗死后内源性HMGB1的释放,并合成重组小鼠HMGB1(recombinant mouse high mobility group box-1 protein,rm HMGB1)蛋白进行体外实验研究。选择心梗后参与疾病病程调控的3种主要心脏组成细胞类型心肌细胞系H9C2、成纤维细胞系10T1/2以及原代巨噬细胞进行研究。构建miR-125b过表达腺病毒及对照腺病毒载体体外感染H9C2和10T1/2细胞及体内感染心梗后心脏组织;合成miR-125b模拟物miR-125b mimic或对照模拟物control mimic转染小鼠原代巨噬细胞;心梗后感染腺病毒的心脏组织予CD11b阳性细胞磁珠分选出巨噬细胞。通过qPCR和ELISA技术检测细胞因子IL-1β、IL-6、IL-12、TNF-a的表达水平。Western blot方法检测炎症反应核因子-κB(nuclear factor-kappa B,NF-κB)和丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路中P65、C-Jun氨基末端激酶(C-Jun N-terminal kinase,JNK)、P38以及细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)的磷酸化水平。结果显示,小鼠心梗早期心肌组织中miR-125b表达增加,同一时期心肌组织检测到,内源性HMGB1释放以及大量炎性细胞因子产生;miR-125b过表达促进心梗后巨噬细胞中生成炎性细胞因子;rm HMGB1重组蛋白可以诱导心肌H9C2细胞、成纤维细胞10T1/2以及原代巨噬细胞中炎性细胞因子IL-1β、IL-6、IL-12、TNF-α表达水平不同程度变化,但是过表达miR-125b仅对rm HMGB1刺激原代巨噬细胞产生炎性细胞因子有选择性正向调控作用,其原因可能与巨噬细胞中P65的磷酸化水平变化有关。该研究结果表明,miR-125b正调控心肌梗死后巨噬细胞中内源性HMGB1诱导的炎症反应,对rm HMGB1诱导心肌细胞及成纤维细胞炎症反应无显著调控作用。
[Abstract]:This study aims to explore the mouse small RNA-125b (microRNA-125b, miR-125b) Regulation after myocardial infarction, endogenous risk factors: high mobility group protein 1 (high mobility group box-1 protein, HMGB1) release induced by inflammatory reaction and comparison of main components in 3 kinds of cells in the regulation of the heart. We established a mouse model of myocardial infarction, myocardial infarction early detection of miR-125b and inflammatory cytokine interleukin -1 beta (interleukin-1 beta, beta IL-1), interleukin -6 (interleukin-6, IL-6), interleukin -12 (interleukin-12, IL-12), tumor necrosis factor alpha (tumor alpha necrosis factor-, TNF-a) the expression changes after myocardial infarction and to observe the effect of overexpression of miR-125b on cardiac function; by immunohistochemical and immunofluorescence method to detect endogenous release of HMGB1 mice after myocardial infarction, and synthesis of recombinant mouse HMGB1 (recombinant mouse high mobility group box-1 protein, RM HMGB1) protein in vitro. 3 main cardiac component cell types, H9C2, 10T1/2 and primary macrophages, were selected to control the disease course after myocardial infarction. Construction of miR-125b over expression of adenovirus and control adenovirus vector and 10T1/2 cells infected with H9C2 in vitro and in vivo infection after myocardial infarction cardiac tissue; synthetic analogue of miR-125b miR-125b mimic or control mimic analog control transfection of mouse primary macrophages; adenovirus infection after myocardial infarction heart tissue to CD11b positive cells sorted out macrophages. The expression levels of cytokine IL-1 beta, IL-6, IL-12 and TNF-a were detected by qPCR and ELISA techniques. Western blot for the detection of inflammatory reaction of nuclear factor kappa B (nuclear factor-kappa B, NF- K B) and mitogen activated protein kinase (mitogen activated protein kinase, MAPK) signal pathway of P65, C-Jun N-terminal kinase (C-Jun N-terminal kinase, JNK, P38) and extracellular signal regulated kinase extracellular (signal-regulated kinase, ERK) phosphorylation. The results showed that the increased expression of miR-125b in mice myocardium in myocardial infarction, myocardial tissue was detected during the same period, the release of endogenous HMGB1 and produce large amounts of inflammatory cytokines; miR-125b overexpression promotes macrophage after myocardial infarction produce inflammatory cytokines; RM HMGB1 recombinant protein H9C2 can induce myocardial cells, fibroblasts and primary 10T1/2 macrophage inflammatory cytokines IL-1, IL-6, IL-12, TNF- beta alpha expression level changes, but only the RM expression of miR-125b HMGB1 stimulated primary macrophages to produce inflammatory cytokines with selective positive regulation effect, the reason may be related to changes in the phosphorylation level of P65 in macrophages. The results indicate that miR-125b is regulating the endogenous HMGB1 induced inflammatory reaction in macrophages after myocardial infarction, and has no significant regulation effect on RM HMGB1 induced inflammatory response in cardiac myocytes and fibroblasts.
【作者单位】：广西医科大学附属肿瘤医院;同济大学附属东方医院;
【基金】：国家自然科学基金(批准号:81373146,81370433) 上海市“创新行动计划”基础研究领域项目(批准号:14JC1405200)资助的课题~~
【分类号】：R542.22
【正文快照】： 心肌梗死(myocardial infarction,MI)是指冠状动脉供血急剧减少或中断,使相应的心肌严重而持续性缺血所致的心肌缺血坏死[1]。心肌梗死会导致梗死闭塞部位的远端心肌持续的坏死,梗死部位与冠状动脉供血区域一致,且多发生于左心室。心梗后,心肌细胞缺血缺氧坏死释放的内源性危

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