本文選題：神經(jīng)病理性疼痛 + 趨化因子MCP-1�。� 參考：《第二軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：單核細(xì)胞趨化蛋白-1 (monocyte chemoattractant protein-1;MCP-1)屬于CC類趨化因子。近年來的研究表明,在神經(jīng)病理性疼痛、炎癥或其他病理性變等情況下,趨化因子MCP-1及其受體CCR2在神經(jīng)元和膠質(zhì)細(xì)胞上表達(dá)上調(diào),并能直接引起痛覺神經(jīng)元興奮性持續(xù)增高、傷害性感受器基因表達(dá)改變。MCP-1被認(rèn)為是疼痛家族的一個新成員,是一個重要的導(dǎo)致疼痛發(fā)生的因子。 辣椒素受體TRPV1在背根神經(jīng)節(jié)、三叉神經(jīng)節(jié)等中小型神經(jīng)元的C類無髓纖維或Aδ神經(jīng)纖維等疼痛感受器上大量表達(dá),是配體門控的非選擇性陽離子通道。越來越多的研究表明,TRPV1在痛覺的產(chǎn)生及痛覺敏感性增強的病理過程中扮演著重要角色。 為了進一步研究MCP-1對TRPV1疼痛效應(yīng)的調(diào)控機制,本課題在大鼠慢性壓迫背根神經(jīng)節(jié)(CCD)的模型上,應(yīng)用Real-time PCR和免疫組織化學(xué)染色明確CCD背根神經(jīng)節(jié)細(xì)胞上,MCP-1受體CCR2與TRPV1等的表達(dá)與分布;用Western blot的方法對CCD、CCI以及SNL三種神經(jīng)損傷模型上CCR2在DRG中的表達(dá)進行了定量分析;應(yīng)用行為學(xué)測試方法觀察MCP-1是否易化CCD大鼠上TRPV1介導(dǎo)的痛覺行為,應(yīng)用全細(xì)胞膜片鉗及鈣離子成像技術(shù),觀察了MCP-1對CCD大鼠DRG神經(jīng)元上TRPV1通道的調(diào)控作用及可能的細(xì)胞內(nèi)信號途徑。主要研究結(jié)果如下; 1應(yīng)用Real-time PCR方法顯示,CCD后TRPV1的mRNA表達(dá)增加并應(yīng)用免疫組化方法顯示CCD后TRPV1表達(dá)增加,用Western blot的方法對CCD、CCI以及SNL三種神經(jīng)損傷模型上CCR2在DRG中的表達(dá)進行了定量分析,結(jié)果表明,三種損傷方式都能引起CCR2的表達(dá)量增加,尤以CCD損傷后CCR2增加特別明顯,在DRG組織切片和急性分離的DRG神經(jīng)元上用免疫熒光方法顯示CCR2和TRPV1共表達(dá)于CCD神經(jīng)元,這就為MCP-1對TRPV1的調(diào)控作用提供形態(tài)學(xué)基礎(chǔ)。 2動物自發(fā)痛反應(yīng)行為測試結(jié)果顯示,MCP-1增強Capsaicin誘發(fā)的疼痛行為,與單獨給與MCP-1和Capsaicin有顯著性差異。 3全細(xì)胞膜片鉗記錄顯示,MCP-1增強在急性分離的CCD神經(jīng)元上TRPV1受體激動劑Capsaicin誘導(dǎo)的膜電位去極化幅度及內(nèi)向電流幅度。應(yīng)用Ca~(2+)成像技術(shù)觀察對急性分離的CCD神經(jīng)元在MCP-1作用前后對Capsaicin誘發(fā)的細(xì)胞內(nèi)游離鈣離子的反應(yīng)。結(jié)果表明,MCP-1(100 ng/ml)增強Capsaicin誘發(fā)的細(xì)胞內(nèi)鈣熒光強度 4在急性分離的CCD神經(jīng)元上,應(yīng)用Ca~(2+)成像技術(shù)觀察PLC阻斷劑U73122對MCP-1增強Capsaicin誘發(fā)的細(xì)胞內(nèi)鈣熒光強度的調(diào)控作用。結(jié)果顯示U73122有效地阻斷MCP-1對TRPV1的增強效應(yīng)。動物自發(fā)痛反應(yīng)行為測試結(jié)果顯示,PLC阻斷劑U73122有效地阻斷MCP-1對Capsaicin引發(fā)的自發(fā)痛的易化作用。 綜上所述,提示CCD損傷后DRG神經(jīng)元上調(diào)表達(dá)的CCR2,在MCP-1作用激活后可通過增強TRPV1的功能,使痛覺神經(jīng)元的興奮性提高,從而參與痛覺過敏的產(chǎn)生。
[Abstract]:Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine. Recent studies have shown that in the presence of neuropathic pain, inflammation or other pathological changes, the expression of chemokine MCP-1 and its receptor CCR2 is up-regulated in neurons and glial cells, and can directly induce a sustained increase in the excitability of pain neurons. Changes in gene expression of nociceptors. MCP-1 is considered to be a new member of the pain family and an important factor leading to pain. Capsaicin receptor TRPV1 is highly expressed in pain receptors such as class C unmyelinated fibers or A 未 nerve fibers in small and medium-sized neurons such as dorsal root ganglion, trigeminal ganglion and so on. It is a ligand gated non-selective cationic channel. More and more studies have shown that TRPV1 plays an important role in the generation of pain and the enhancement of pain sensitivity. In order to further study the mechanism of MCP-1 regulating the pain effect of TRPV1, the model of chronic compression of dorsal root ganglion (DRG) in rats was studied. The expression and distribution of MCP-1 receptor CCR2 and TRPV1 on CCD dorsal root ganglion cells were determined by Real-time PCR and immunohistochemical staining, and the expression of CCR2 in DRG was quantitatively analyzed by Western blot method. Behavioral test was used to observe whether MCP-1 facilitated the pain induced by TRPV1 on CCD rats. Whole cell patch-clamp and calcium imaging were used. The effect of MCP-1 on TRPV1 channel in DRG neurons of CCD rats and the possible intracellular signal pathway were observed. The main results are as follows; 1 the expression of mRNA in TRPV1 was increased by Real-time PCR method and the expression of TRPV1 in CCD was detected by immunohistochemistry. The expression of CCR2 in DRG was quantitatively analyzed by Western blot method. The expression of CCR2 was increased in all three kinds of injury modes, especially CCR2 after CCD injury. CCR2 and TRPV1 co-expressed in CCD neurons by immunofluorescence method in DRG tissue sections and acute isolated DRG neurons. This provides a morphological basis for the regulation of TRPV1 by MCP-1. 2 the results of spontaneous pain response test showed that MCP-1 enhanced the pain behavior induced by Capsaicin, which was significantly different from that of MCP-1 and Capsaicin alone. 3 whole cell patch clamp records showed that MCP-1 enhanced the depolarization amplitude and inward current amplitude induced by TRPV1 receptor agonist Capsaicin on the acutely isolated CCD neurons. Ca~(2 imaging was used to observe the response of acutely isolated CCD neurons to intracellular free calcium ions induced by Capsaicin before and after MCP-1 treatment. The results showed that MCP-1 + 100ng / ml enhanced intracellular calcium fluorescence induced by Capsaicin. 4 in acute isolated CCD neurons, the effect of U73122, a PLC blocker, on the intracellular calcium fluorescence intensity induced by Capsaicin induced by MCP-1 was observed by Ca~(2 imaging technique. The results showed that U73122 effectively blocked the enhancement effect of MCP-1 on TRPV1. The results of animal spontaneous pain response test showed that U73122 could effectively block the facilitation of Capsaicin induced spontaneous pain by MCP-1. In conclusion, it is suggested that the up-regulated expression of CCR2 in DRG neurons after CCD injury can increase the excitability of pain neurons by enhancing the function of TRPV1 after the activation of MCP-1, and thus participate in the production of hyperalgesia.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

【引證文獻】

相關(guān)期刊論文 前1條

1 汪洋;王家林;;趨化因子配體及其抗體對骨癌痛患者脊髓膠質(zhì)細(xì)胞活化的影響及與骨癌痛的相關(guān)性[J];中國老年學(xué)雜志;2013年02期

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本文編號：1836272