【摘要】：背景：偏頭痛是一種嚴(yán)重的腦功能紊亂性疾病,其全球患病率在12%左右,嚴(yán)重影響患者的日常工作和生活。其臨床表現(xiàn)為單側(cè)的搏動性頭痛,日�；顒涌杉觿√弁闯潭�,常伴有惡心、嘔吐等癥狀。三叉神經(jīng)血管系統(tǒng)(trigemino vascular system, TGVS)的激活是偏頭痛發(fā)生發(fā)展過程中最重要的事件,在此過程中三叉神經(jīng)末梢釋放血管活性神經(jīng)肽類物質(zhì),后者導(dǎo)致硬腦膜上發(fā)生神經(jīng)源性炎癥,從而使硬腦膜傷害性感受器長時程敏化,臨床表現(xiàn)為嚴(yán)重的偏側(cè)頭痛,現(xiàn)在認(rèn)為這種現(xiàn)象系初級痛覺神經(jīng)元發(fā)生了周圍敏化。炎性小體系組織細(xì)胞在受到內(nèi)外源性刺激時組裝合成的一類多蛋白復(fù)合體,能夠促進(jìn)細(xì)胞因子IL-1p和IL-18的成熟和分泌。成熟的IL-1β通過激活三叉神經(jīng)節(jié)衛(wèi)星膠質(zhì)細(xì)胞,引起一系列炎癥反應(yīng),進(jìn)而導(dǎo)致偏頭痛外周敏化的發(fā)生。炎性小體有4種類型:NALP1,NALP3, NLRC4和AIM2。其中,NALP3炎性小體是最重要的一種類型。研究已經(jīng)證實激活炎性小體主要通過P2X7受體、活性氧及組織蛋白酶三條通路。但是目前罕有關(guān)于NALP3炎性小體參與偏頭痛外周敏化的發(fā)生及其激活途徑方面的研究。因此我們通過建立炎性刺激大鼠硬腦膜模型(常用的偏頭痛模型)提出假設(shè)：炎性刺激大鼠硬腦膜模型中可以觀察到活化的NALP3炎性小體,此外,P2X7受體可能參與其激活。 目的：通過采用炎性刺激大鼠硬腦膜疼痛模型,探討嘌呤受體P2X配體門控性離子通道7(P2X7R)是否參與偏頭痛發(fā)作過程中三叉神經(jīng)節(jié)內(nèi)NALP3炎性小體的激活。 方法：健康成年雄性SD大鼠42只,免疫印跡實驗取27只隨機(jī)分為9組,每組3只,分別為空白對照組、致炎劑3h組、致炎劑6h組、致炎劑1d組、致炎劑2d組、致炎劑3d組、生理鹽水對照組、致炎劑2d對照組和亮藍(lán)G(BBG)治療組。免疫熒光雙重染色實驗取15只隨機(jī)分為3組,每組5只,分別為空白對照組、生理鹽水對照組和致炎劑2d組。觀察各組大鼠P2X7R和NALP3在三叉神經(jīng)節(jié)中的表達(dá)情況,并且運用P2X7受體特異性拮抗劑亮藍(lán)G(Brilliant Blue G, BBG)干預(yù)后,觀察各組大鼠P2X7R. NALP3的表達(dá)。 結(jié)果：①免疫熒光雙重染色結(jié)果：NALP3蛋白定位表達(dá)于三叉神經(jīng)元細(xì)胞內(nèi),而P2X7R主要表達(dá)于衛(wèi)星膠質(zhì)細(xì)胞。致炎劑組NALP3及P2X7R熒光強(qiáng)度明顯高于空白對照組和生理鹽水對照組,說明致炎劑刺激大鼠硬腦膜能成功誘導(dǎo)三叉神經(jīng)節(jié)中NALP3及P2X7R蛋白的表達(dá)。②免疫印跡結(jié)果：與空白對照組相比,各致炎劑組NALP3、P2X7R蛋白表達(dá)量均明顯升高,具有統(tǒng)計學(xué)意義(P0.05)；各蛋白表達(dá)量隨給藥次數(shù)及藥物作用時間基本呈正相關(guān)。BBG能有效抑制P2X7R和NALP3的蛋白表達(dá),與空白組和生理鹽水對照組相比,差異具有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：在炎性刺激大鼠硬腦膜模型中可以觀察到三叉神經(jīng)節(jié)內(nèi)P2X7R和NALP3炎性小體的激活,此外,P2X7R特異性拮抗劑BBG能有效地抑制NALP3蛋白的表達(dá),說明P2X7受體參與炎性小體的激活。
[Abstract]:Background: migraine is a severe disorder of brain function. The global prevalence rate of migraine is about 12%, which seriously affects the daily work and life of patients. Its clinical manifestation is unilateral pulsatile headache, daily activities can aggravate the degree of pain, often accompanied by nausea, vomiting and other symptoms. The activation of (trigemino vascular system, TGVS) in the trigeminal vascular system is the most important event in the occurrence and development of migraine, during which the trigeminal nerve endings release vasoactive neuropeptides, which lead to neurogenic inflammation on the dura mater. So that the dura nociceptive receptors were sensitized for a long time, and the clinical manifestations were severe hemiheadache. It is now believed that this phenomenon is caused by peripheral sensitization of primary nociceptive neurons. A class of polyprotein complexes assembled by inflammatory small tissue cells stimulated by exogenous and exogenous stimuli can promote the maturation and secretion of cytokines IL-1p and IL-18. Mature IL-1 尾 activates satellite glial cells in the trigeminal ganglion, causing a series of inflammatory reactions, which leads to peripheral sensitization of migraine. Inflammatory corpuscles have four types: NALP1, NALP3, NLRC4 and AIM2. The inflammatory corpuscles of NALP3 are the most important type. It has been demonstrated that the activation of inflammatory corpuscles is mainly via three pathways: P2X7 receptor, reactive oxygen species and cathepsin. However, there are few studies on the role of NALP3 inflammatory corpuscles in the pathogenesis and activation of peripheral sensitization of migraine. Therefore, we hypothesized that activated NALP3 inflammatory corpuscles can be observed in the inflammatory stimulation rat dural model (the commonly used migraine model), in addition, P2X7 receptor may be involved in its activation. Aim: to investigate whether purine receptor P2X ligand gated ion channel 7 (P2X7R) is involved in the activation of NALP3 inflammatory corpuscles in trigeminal ganglion during migraine attack. Methods: Forty-two healthy adult male SD rats were randomly divided into 9 groups with 3 rats in each group: the blank control group, the inflammatory agent group for 3 hours, the inflammatory agent group for 6 hours, the inflammatory agent group for 1 day, the inflammatory agent group for 2 days, and the inflammatory agent group for 3 days. Normal saline control group, inflammatory agent 2 days control group and bright blue G (BBG) treatment group. Fifteen rats were randomly divided into 3 groups: blank control group, normal saline control group and inflammatory agent group for 2 days. The expression of P2X7R and NALP3 in trigeminal ganglion was observed, and P2X7R was observed after treated with brilliant blue G (Brilliant Blue G, BBG), a specific antagonist of P2X7 receptor. Expression of NALP3. Results the results of double immunofluorescence staining showed that the protein was localized in trigeminal neurons, while P2X7R was mainly expressed in satellite glial cells. The fluorescence intensity of NALP3 and P2X7R in the inflammatory group was significantly higher than that in the blank control group and the saline control group. The results showed that the expression of NALP3 and P2X7R protein in trigeminal ganglion could be induced successfully by stimulation of dural with inflammatory agent. The results showed that compared with the blank control group, the expression of NALP3,P2X7R protein in each group was significantly increased (P0.05). The expression of each protein was positively correlated with the times of administration and the time of drug action. BBG could effectively inhibit the protein expression of P2X7R and NALP3 compared with the blank group and normal saline control group the difference was statistically significant (P0.05). Conclusion: the activation of P2X7R and NALP3 inflammatory corpuscles in trigeminal ganglion can be observed in the rat dural model of inflammatory stimulation. In addition, BBG, a specific antagonist of P2X7R, can effectively inhibit the expression of NALP3 protein, indicating that P2X7 receptor is involved in the activation of inflammatory corpuscles.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R747.2

【二級參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉婷;沈飛飛;韋俊超;萬琪;;頭痛寧對電刺激三叉神經(jīng)節(jié)大鼠腦膜血流與CGRP的影響[J];中國疼痛醫(yī)學(xué)雜志;2010年04期

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本文編號：2219341