本文關(guān)鍵詞：microRNA-125b調(diào)控心肌梗死后炎癥的作用及其在不同心臟組成細(xì)胞中的調(diào)控作用　出處：《中國細(xì)胞生物學(xué)學(xué)報(bào)》2016年12期 　論文類型：期刊論文

  更多相關(guān)文章： miR-b 心肌梗死 炎癥反應(yīng) HMGB 巨噬細(xì)胞

【摘要】：該研究旨在探究小鼠微小RNA-125b(microRNA-125b,miR-125b)調(diào)控心肌梗死后,內(nèi)源性危險(xiǎn)因子即高遷移率族蛋白1(high mobility group box-1 protein,HMGB1)釋放誘導(dǎo)的炎癥反應(yīng)及其在3種心臟主要組成細(xì)胞中調(diào)控作用的比較。首先建立小鼠心梗模型,檢測心梗早期miR-125b及炎性細(xì)胞因子白細(xì)胞介素-1β(interleukin-1β,IL-1β)、白細(xì)胞介素-6(interleukin-6,IL-6)、白細(xì)胞介素-12(interleukin-12,IL-12)、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-a)的表達(dá)變化并觀察心梗后miR-125b過表達(dá)對心功能的影響;用免疫組織化學(xué)和免疫熒光方法檢測小鼠心肌梗死后內(nèi)源性HMGB1的釋放,并合成重組小鼠HMGB1(recombinant mouse high mobility group box-1 protein,rm HMGB1)蛋白進(jìn)行體外實(shí)驗(yàn)研究。選擇心梗后參與疾病病程調(diào)控的3種主要心臟組成細(xì)胞類型心肌細(xì)胞系H9C2、成纖維細(xì)胞系10T1/2以及原代巨噬細(xì)胞進(jìn)行研究。構(gòu)建miR-125b過表達(dá)腺病毒及對照腺病毒載體體外感染H9C2和10T1/2細(xì)胞及體內(nèi)感染心梗后心臟組織;合成miR-125b模擬物miR-125b mimic或?qū)φ漳M物control mimic轉(zhuǎn)染小鼠原代巨噬細(xì)胞;心梗后感染腺病毒的心臟組織予CD11b陽性細(xì)胞磁珠分選出巨噬細(xì)胞。通過qPCR和ELISA技術(shù)檢測細(xì)胞因子IL-1β、IL-6、IL-12、TNF-a的表達(dá)水平。Western blot方法檢測炎癥反應(yīng)核因子-κB(nuclear factor-kappa B,NF-κB)和絲裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信號(hào)通路中P65、C-Jun氨基末端激酶(C-Jun N-terminal kinase,JNK)、P38以及細(xì)胞外信號(hào)調(diào)節(jié)激酶(extracellular signal-regulated kinase,ERK)的磷酸化水平。結(jié)果顯示,小鼠心梗早期心肌組織中miR-125b表達(dá)增加,同一時(shí)期心肌組織檢測到,內(nèi)源性HMGB1釋放以及大量炎性細(xì)胞因子產(chǎn)生;miR-125b過表達(dá)促進(jìn)心梗后巨噬細(xì)胞中生成炎性細(xì)胞因子;rm HMGB1重組蛋白可以誘導(dǎo)心肌H9C2細(xì)胞、成纖維細(xì)胞10T1/2以及原代巨噬細(xì)胞中炎性細(xì)胞因子IL-1β、IL-6、IL-12、TNF-α表達(dá)水平不同程度變化,但是過表達(dá)miR-125b僅對rm HMGB1刺激原代巨噬細(xì)胞產(chǎn)生炎性細(xì)胞因子有選擇性正向調(diào)控作用,其原因可能與巨噬細(xì)胞中P65的磷酸化水平變化有關(guān)。該研究結(jié)果表明,miR-125b正調(diào)控心肌梗死后巨噬細(xì)胞中內(nèi)源性HMGB1誘導(dǎo)的炎癥反應(yīng),對rm HMGB1誘導(dǎo)心肌細(xì)胞及成纖維細(xì)胞炎癥反應(yīng)無顯著調(diào)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.
【作者單位】： 廣西醫(yī)科大學(xué)附屬腫瘤醫(yī)院;同濟(jì)大學(xué)附屬東方醫(yī)院;
【基金】：國家自然科學(xué)基金(批準(zhǔn)號(hào):81373146,81370433) 上海市“創(chuàng)新行動(dòng)計(jì)劃”基礎(chǔ)研究領(lǐng)域項(xiàng)目(批準(zhǔn)號(hào):14JC1405200)資助的課題~~
【分類號(hào)】：R542.22
【正文快照】： 心肌梗死(myocardial infarction,MI)是指冠狀動(dòng)脈供血急劇減少或中斷,使相應(yīng)的心肌嚴(yán)重而持續(xù)性缺血所致的心肌缺血壞死[1]。心肌梗死會(huì)導(dǎo)致梗死閉塞部位的遠(yuǎn)端心肌持續(xù)的壞死,梗死部位與冠狀動(dòng)脈供血區(qū)域一致,且多發(fā)生于左心室。心梗后,心肌細(xì)胞缺血缺氧壞死釋放的內(nèi)源性危

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