【摘要】：[研究背景及目的]炎性疼痛普遍存在于臨床情況中,也是困擾患者的主要慢性疼痛類型。機(jī)制研究發(fā)現(xiàn),小膠質(zhì)細(xì)胞及其分泌的促炎癥因子參與炎性疼痛的發(fā)生與長(zhǎng)期維持。而近年來(lái)的研究揭示,miR-146a對(duì)炎癥反應(yīng)有負(fù)向調(diào)節(jié)作用,且很可能也參與了疼痛機(jī)制。本研究的目的是希望通過人為干預(yù),在體外和體內(nèi)實(shí)驗(yàn)中,高表達(dá)miR-146a,驗(yàn)證它能否減少小膠質(zhì)細(xì)胞激活后及大鼠炎性疼痛模型中脊髓的促炎癥因子表達(dá),并能否緩解大鼠炎性疼痛。[研究方法]體外——PCR法檢測(cè)細(xì)菌脂多糖(LPS)刺激小膠質(zhì)細(xì)胞株(BV2細(xì)胞)后的miR-146a表達(dá)水平；在miR-146a模擬物轉(zhuǎn)染BV2細(xì)胞后,采用ELISA法檢測(cè)細(xì)胞上清液中的TNFa和IL-6表達(dá)情況；同時(shí)分別采用PCR和western-blot檢測(cè)細(xì)胞中IRAK1和TRAF6的基因和蛋白表達(dá)情況。體內(nèi)——采用完全氟氏佐劑(CFA)制備大鼠慢性炎性疼痛模型,鞘內(nèi)注射miR-146a模擬物進(jìn)行中樞神經(jīng)系統(tǒng)內(nèi)轉(zhuǎn)染；采用機(jī)械和熱痛刺激進(jìn)行疼痛行為學(xué)檢測(cè)；提取腰段脊髓RNA,采用實(shí)時(shí)熒光定量PCR檢測(cè)中樞神經(jīng)系統(tǒng)內(nèi)miR-146a、TLR4、IRAK1、TRAF6、TNFa和IL-6表達(dá)變化。[研究結(jié)果]體外——與正常組或陰性對(duì)照組比較,采用10nM、50nM、100nM三個(gè)轉(zhuǎn)染濃度對(duì)BV2細(xì)胞進(jìn)行miR-146a模擬物轉(zhuǎn)染,均可明顯增加細(xì)胞內(nèi)miR-146a含量；其中以50nM轉(zhuǎn)染濃度效率最佳；LPS刺激導(dǎo)致BV2細(xì)胞激活,可以明顯增加細(xì)胞內(nèi)IRAK1和TRAF6表達(dá),也可以明顯誘發(fā)細(xì)胞分泌更多的TNFa和IL-6;用miR-146a模擬物轉(zhuǎn)染可以明顯降低TNFa和IL-6的表達(dá)水平,同時(shí)明顯降低細(xì)胞內(nèi)TRAF6,而非IRAK1的表達(dá)。體內(nèi)——在CFA誘發(fā)的大鼠炎性疼痛中,miR-146a出現(xiàn)異常表達(dá),在CFA注射后第1天有所降低,在第3天顯著升高,第7天有所回落,但仍然顯著高于對(duì)照組；而中樞神經(jīng)系統(tǒng)內(nèi)的TLR4、IRAK1、 TRAF6、TNFa和IL-6隨著時(shí)間逐漸升高；鞘內(nèi)注射miR-146a模擬物可以明顯緩解大鼠的疼痛,其機(jī)械縮足閾值和熱縮足潛伏期均有所回升；PCR分析證實(shí)miR-146a模擬物鞘內(nèi)注射可以顯著增加脊髓內(nèi)miR-146a含量,同時(shí)可以降低促炎癥因子TNFa口IL-6的表達(dá)水平,并且TLR4信號(hào)通路上的IRAK1和TRAF6分子的表達(dá)亦有明顯降低。[研究結(jié)論]1) miR-146a模擬物的體外和體內(nèi)轉(zhuǎn)染,都可以有效提高細(xì)胞或組織內(nèi)的miR-146a含量；2) miR-146a可以減少LPS導(dǎo)致的BV2細(xì)胞促炎癥因子分泌,并且該作用可能是通過負(fù)反饋調(diào)節(jié)TLR4信號(hào)通路上的TRAF6分子而進(jìn)行；3)miR-146a可以減輕CFA誘導(dǎo)的大鼠炎性疼痛,很有可能參與了慢性疼痛的發(fā)展、維持及調(diào)節(jié)。
[Abstract]:Background and objective: inflammatory pain is common in clinical conditions and is the main chronic pain type in patients. It is found that microglia and its proinflammatory cytokines are involved in the occurrence and long-term maintenance of inflammatory pain. Recent studies have revealed that miR-146a plays a negative role in inflammatory response and may also be involved in the pain mechanism. The aim of this study is to investigate whether the overexpression of miR-146a, can reduce the expression of pro-inflammatory factors in spinal cord after activation of microglia and in rat inflammatory pain model by human intervention in vitro and in vivo. And can alleviate the inflammatory pain in rats. [methods] the expression of miR-146a in microglia (BV2 cells) stimulated by lipopolysaccharide (LPS) was detected by PCR method in vitro. After transfection of miR-146a mimics into BV2 cells, the expression of TNFa and IL-6 in supernatant was detected by ELISA assay, and the gene and protein expression of IRAK1 and TRAF6 were detected by PCR and western-blot, respectively. In vivo-chronic inflammatory pain model of rats was established by complete fluorine adjuvant (CFA), intrathecal injection of miR-146a mimics for central nervous system transfection, mechanical and thermal pain stimulation for pain behavior detection; The expression of miR-146a,TLR4,IRAK1,TRAF6,TNFa and IL-6 in central nervous system (CNS) was detected by real-time fluorescence quantitative PCR (PCR). [results] compared with the normal group or the negative control group in vitro, the miR-146a content in BV2 cells was significantly increased by transfection of miR-146a mimics with 10 nm M ~ (50) nM ~ (10) nM ~ (100) nm. The concentration efficiency of 50nM transfection was the best. LPS stimulation led to activation of BV2 cells, which significantly increased the expression of IRAK1 and TRAF6, and induced more TNFa and IL-6; secretion. Transfection with miR-146a mimics could significantly reduce the expression of TNFa and IL-6, and TRAF6, instead of IRAK1 in cells. In vivo-in CFA induced inflammatory pain in rats, the expression of miR-146a was abnormal, decreased on the first day after CFA injection, increased significantly on the third day, and decreased on the 7th day, but still significantly higher than that of the control group. However, TLR4,IRAK1, TRAF6,TNFa and IL-6 in the central nervous system increased gradually with time, and intrathecal injection of miR-146a mimics significantly alleviated the pain in rats, and the mechanical foot contraction threshold and the latent period of thermal contraction increased. PCR analysis showed that intrathecal injection of miR-146a mimics could significantly increase the content of miR-146a in the spinal cord and decrease the expression of IL-6 in the mouth of TNFa, and the expression of IRAK1 and TRAF6 in TLR4 signaling pathway. [conclusion] 1) transfection of miR-146a mimics in vitro and in vivo can effectively increase the content of miR-146a in cells or tissues; 2) miR-146a can reduce the secretion of inflammatory cytokines induced by LPS in BV2 cells, and this effect may be mediated by negative feedback regulation of TRAF6 molecules in TLR4 signaling pathway. 3) miR-146a can alleviate the inflammatory pain induced by CFA in rats, which may be involved in the development, maintenance and regulation of chronic pain.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R402

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本文編號(hào)：2384739