本文選題：NMDA + NMDA受體　； 參考：《河北醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：目前，病理性疼痛的產(chǎn)生機(jī)制已經(jīng)從單獨(dú)的神經(jīng)元機(jī)制，轉(zhuǎn)向了神經(jīng)元與膠質(zhì)細(xì)胞相互作用機(jī)制，尤其是小膠質(zhì)細(xì)胞作為中樞神經(jīng)系統(tǒng)定居的吞噬細(xì)胞和主要免疫反應(yīng)細(xì)胞受到了廣泛的關(guān)注。大量研究表明，在外周神經(jīng)損傷和傷害性刺激后，脊髓后角的小膠質(zhì)細(xì)胞被激活，合成和分泌多種細(xì)胞因子和趨化因子包括白介素(IL-1)、腫瘤壞死因子（TNF-a）、前列腺素（PGE2）和一氧化氮（NO）等，促進(jìn)病理性疼痛和痛覺(jué)敏感的產(chǎn)生。給予小膠質(zhì)細(xì)胞功能或代謝的抑制藥物如米諾環(huán)素或一些受體的抑制劑，則可明顯地對(duì)病理性疼痛和痛覺(jué)過(guò)敏產(chǎn)生抑制作用。這些研究結(jié)果表明脊髓小膠質(zhì)細(xì)胞的活化在調(diào)節(jié)病理性疼痛和痛覺(jué)過(guò)敏的過(guò)程中發(fā)揮重要作用。然而，病理性疼痛過(guò)程中脊髓小膠質(zhì)細(xì)胞活化機(jī)制仍然不十分清楚。 N-甲基-D-天門(mén)冬氨酸（N-methyl-D-aspartate，NMDA）受體是興奮性氨基酸受體的一種亞型，屬于離子型受體，廣泛分布于中樞神經(jīng)系統(tǒng)，在脊髓的小膠質(zhì)細(xì)胞上也有分布。生理?xiàng)l件下，該受體參與調(diào)節(jié)神經(jīng)系統(tǒng)生長(zhǎng)、發(fā)育，學(xué)習(xí)、記憶等復(fù)雜的生理功能。病理情況下，激活的NMDA受體引起細(xì)胞內(nèi)一系列級(jí)聯(lián)放大效應(yīng)，增強(qiáng)突觸前和突出后神經(jīng)元的興奮性，從而參與介導(dǎo)傷害性信息的傳遞與整合，在病理性疼痛和痛覺(jué)過(guò)敏形成過(guò)程中發(fā)揮重要作用。 我室以往應(yīng)用免疫印跡分析和免疫組化技術(shù)，觀察到椎管內(nèi)注射N(xiāo)MDA受體非競(jìng)爭(zhēng)性拮抗劑MK-801可抑制蜜蜂毒疼痛模型引起的脊髓后角小膠質(zhì)細(xì)胞的活化，初步證明NMDA受體在引起脊髓后角小膠質(zhì)細(xì)胞激活中的作用。 本實(shí)驗(yàn)則采用免疫組化技術(shù)，觀察椎管內(nèi)給予NMDA受體選擇性激動(dòng)劑NMDA對(duì)脊髓小膠質(zhì)細(xì)胞激活的影響，進(jìn)一步研究NMDA及其受體在傷害性信息傳入所致脊髓后角小膠質(zhì)細(xì)胞激活中的作用，為闡明病理性疼痛及痛覺(jué)過(guò)敏過(guò)程中脊髓小膠質(zhì)細(xì)胞激活的機(jī)制提供實(shí)驗(yàn)依據(jù)。 方法：雄性Sprague-Dawley大鼠95只，體重260-280g，隨機(jī)分為以下各組(n=5)。 sham組：椎管內(nèi)注射生理鹽水10μl，于相應(yīng)的時(shí)間點(diǎn)測(cè)定熱輻射縮足潛伏期、熱甩尾潛伏期和機(jī)械刺激縮足閾值后，取腰5脊髓節(jié)段，觀察脊髓后角小膠質(zhì)細(xì)胞CD11b/c（OX-42）的表達(dá)。 NMDA I組：椎管內(nèi)注射N(xiāo)MDA溶液10μl （10nmol），分別于注射后1h、4h、8h、12h、1d、2d和3d測(cè)定熱輻射縮足潛伏期、熱甩尾潛伏期和機(jī)械刺激縮足閾值后，取腰5脊髓節(jié)段，觀察脊髓后角小膠質(zhì)細(xì)胞CD11b/c（OX-42）表達(dá)的時(shí)間變化特點(diǎn)，分析CD11b/c（OX-42）的表達(dá)與痛覺(jué)過(guò)敏的時(shí)間對(duì)應(yīng)關(guān)系。 NMDA II組：椎管內(nèi)注射N(xiāo)MDA溶液10μl。根據(jù)NMDA的劑量，進(jìn)一步分為0.1nmol，1nmol和10nmol三個(gè)亞組。每組動(dòng)物于椎管內(nèi)注射后8h測(cè)定熱輻射縮足潛伏期、熱甩尾潛伏期和機(jī)械刺激縮足閾值后，取腰5脊髓節(jié)段，觀察NMDA上調(diào)脊髓后角小膠質(zhì)細(xì)胞CD11b/（cOX-42）的表達(dá)和導(dǎo)致痛覺(jué)過(guò)敏的劑量依賴性。 MK-801+NMDA組：首先椎管內(nèi)注射N(xiāo)MDA受體抑制劑MK-801溶液10μl (50nmol)，15min后椎管內(nèi)注射N(xiāo)MDA溶液10μl (10nmol)，注射N(xiāo)MDA8h測(cè)定熱輻射縮足潛伏期、熱甩尾潛伏期和機(jī)械刺激縮足閾值后，取腰5脊髓節(jié)段，觀察MK-801對(duì)NMDA引起的脊髓后角小膠質(zhì)細(xì)胞CD11b/c（OX-42）表達(dá)的上調(diào)和痛覺(jué)過(guò)敏的影響。 以上各組動(dòng)物均于相應(yīng)時(shí)間點(diǎn)多聚甲醛灌注固定，，取腰5（L5）脊髓節(jié)段，冰凍連續(xù)切片，免疫組化法觀察L5脊髓后角CD11b/c（OX-42）表達(dá)，反映脊髓后角小膠質(zhì)細(xì)胞激活的狀態(tài)。 數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（x±s）表示。應(yīng)用SPSS統(tǒng)計(jì)軟件13.0對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行單因素方差分析（One-WayANOVA），比較組間差異。有顯著差異者用最小顯著差法進(jìn)行兩兩比較，以p0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1痛行為反應(yīng) 1.1自發(fā)痛行為反應(yīng)椎管內(nèi)注射N(xiāo)MDA后大鼠可出現(xiàn)緊張性的咬、舔后足或尾、肢體扭動(dòng)和吱叫等特征性的傷害性行為反應(yīng)，而椎管內(nèi)預(yù)先注射MK-801（MK-801+NMDA組）則可抑制上述表現(xiàn)。 1.2熱縮足和熱甩尾潛伏期在NMDA I組，椎管內(nèi)注射N(xiāo)MDA（10nmol）后1h，大鼠的熱縮足潛伏期開(kāi)始縮短（p0.05），可持續(xù)到12h。熱縮足潛伏期1d時(shí)恢復(fù)到sham水平，表明椎管內(nèi)注射N(xiāo)MDA后早期就可出現(xiàn)熱痛覺(jué)過(guò)敏，并且這種現(xiàn)象可持續(xù)一段時(shí)間。在NMDAII組，NMDA組的熱縮足潛伏期縮短呈現(xiàn)一定的劑量依賴關(guān)系，表現(xiàn)為隨著NMDA注射劑量的增大，熱縮足潛伏期的縮短逐漸明顯。MK-801+NMDA組與相同劑量NMDA組相比，熱縮足潛伏期顯著延長(zhǎng)（p0.05），表明椎管內(nèi)注射MK-801可抑制上述熱痛覺(jué)過(guò)敏。熱甩尾潛伏期的變化特點(diǎn)與熱縮足潛伏期相似。 1.3機(jī)械縮足反射閾值在NMDA I組，大鼠椎管內(nèi)注射N(xiāo)MDA后4h機(jī)械縮足反射閾值開(kāi)始出現(xiàn)降低（p0.05），可延長(zhǎng)至椎管內(nèi)注射N(xiāo)MDA后1d。機(jī)械縮足反射閾值2d時(shí)恢復(fù)到sham水平，表明椎管內(nèi)注射N(xiāo)MDA后早期也可以表現(xiàn)出機(jī)械痛覺(jué)過(guò)敏，并且這種表現(xiàn)可持續(xù)較長(zhǎng)時(shí)間。在NMDA II組，NMDA組的機(jī)械縮足反射閾值降低呈現(xiàn)一定的劑量依賴關(guān)系，表現(xiàn)為隨著NMDA注射劑量的增大，機(jī)械縮足反射閾值減小逐漸明顯。MK-801+NMDA組與相同劑量NMDA組相比，機(jī)械縮足反射閾值明顯增高（p0.05），表明椎管內(nèi)注射MK-801可抑制上述機(jī)械痛覺(jué)過(guò)敏。 2脊髓后角CD11b/c（OX-42）表達(dá)變化與sham組相比，椎管內(nèi)注射N(xiāo)MDA4h后，脊髓后角小膠質(zhì)細(xì)胞CD11b/c（OX-42）的表達(dá)明顯升高（p0.05），鏡下觀察小膠質(zhì)細(xì)胞被激活，其特征為胞體明顯變大，突起增粗變短，染色加深。上述CD11b/c（OX-42）的表達(dá)和小膠質(zhì)細(xì)胞的形態(tài)變化在注射N(xiāo)MDA8h后達(dá)到高峰，1d后開(kāi)始減弱，3d基本恢復(fù)到sham水平。隨著大鼠椎管內(nèi)給予NMDA劑量的增加（0.1nmol,1nmol和10nmol），脊髓后角CD11b/c（OX-42）表達(dá)逐漸升高，呈明顯的劑量依賴性。預(yù)先椎管內(nèi)注射N(xiāo)MDA受體阻斷劑MK-801可顯著抑制上述NMDA引起的CD11b/c（OX-42）表達(dá)的上調(diào)（p0.05）。 結(jié)論： 1椎管內(nèi)注射N(xiāo)MDA受體特異性激動(dòng)劑NMDA可以引起傷害性感受和痛覺(jué)過(guò)敏，這種傷害性行為可被NMDA受體非競(jìng)爭(zhēng)性抑制劑MK-801在一定程度上所抑制。 2椎管內(nèi)注射N(xiāo)MDA受體特異性激動(dòng)劑NMDA可以引起脊髓后角小膠質(zhì)細(xì)胞活化，這種變化呈現(xiàn)一定的劑量依賴性，且可被NMDA受體非競(jìng)爭(zhēng)性抑制劑MK-801所抑制。 3這些結(jié)果提示，NMDA及其受體在傷害性信息傳入所致脊髓后角小膠質(zhì)細(xì)胞活化過(guò)程中發(fā)揮重要作用。
[Abstract]:Objective : At present , the mechanism of pathogenesis of pathological pain has shifted from the individual neuron mechanism to the interaction mechanism between neuron and glial cells , especially the phagocytes and main immune response cells settled by microglial cells as the central nervous system .

N - methyl - D - aspartate ( NMDA ) receptor is a subtype of excitatory amino acid receptor , and belongs to ionic receptor . It is widely distributed in the central nervous system and is distributed in the microglial cells of the spinal cord . Under physiological conditions , the receptor is involved in regulating nervous system growth , development , learning , memory and other complex physiological functions .

In the past , we have observed that MK - 801 , a non - competitive antagonist of NMDA receptor in spinal canal , can inhibit the activation of microglial cells in spinal dorsal horn caused by bee venom pain model and demonstrate the role of NMDA receptor in the activation of glial cells after spinal cord injury .

In this experiment , the effects of NMDA receptor selective agonist NMDA on the activation of microglial cells in spinal cord were observed by immunohistochemistry , and the role of NMDA and its receptors in the activation of spinal dorsal horn microglial cells was further studied .

Methods : 95 male Sprague - Dawley rats weighing 260 - 280 g were randomly divided into the following groups ( n = 5 ) .

sham group : 10 渭l of normal saline was injected into the spinal canal , the latent period of heat radiation , the latent period of thermal spin tail and the threshold of mechanical stimulation were measured at the corresponding time point .

In the group of NMDA I , 10 渭l ( 10 nmol ) of NMDA solution was injected into the spinal canal , and the latent period of heat radiation , latent period of heat - spin tail and the threshold of mechanical stimulation were measured at 1h , 4h , 8h , 12h , 1d , 2d and 3d after injection .

NMDA was injected into the spinal canal for 10 渭l . According to the dose of NMDA , the rats were further divided into three subgroups of 0.1 nmol , 1 nmol and 10 nmol . The latency of heat radiation , latency of thermal spin tail and the threshold of mechanical stimulation were measured at 8 h after injection .

MK - 801 + NMDA group : 10 渭l ( 50 nmol ) of NMDA receptor inhibitor MK - 801 was injected into the spinal canal , and 10 渭l ( 10 nmol ) of NMDA solution was injected into the spinal canal after 15min . After injection of NMDA8h , the latent period of heat radiation , the latency of thermal spin tail and the threshold of mechanical stimulation were measured .

5 ( L5 ) spinal cord segments and frozen continuous sections were harvested at the corresponding time points , and the expression of CD11a / c ( OX - 42 ) in L5 spinal cord was observed by immunohistochemical method , which reflected the activation status of microglial cells in the posterior horn of spinal cord .

The data was expressed by mean 鹵 standard deviation ( x 鹵 s ) . SPSS statistical software was applied to analyze the single - factor variance ( One - way ANOVA ) in the experimental data .

Results :

1 Pain response

1.1 After injection of NMDA into the spinal canal by spontaneous pain , the rats were subjected to stress - induced sexual behavior such as tension , licking , hindfoot or tail , limb torsion and squeaking , while MK - 801 ( MK - 801 + NMDA group ) was pre - injected into the spinal canal to inhibit the above expression .

1 . The latent period of heat - shrinkable foot and warm - tail tail in NMDA group was shorter than that in NMDA group ( P < 0.05 ) .

1 . The threshold of mechanical contraction reflex in NMDA group was decreased ( p . 05 ) after injection of NMDA into the spinal canal of NMDA group .

2鑴婇珦鍚庤CD11b/c(OX-42)琛ㄨ揪鍙樺寲涓巗ham緇勭浉姣

本文編號(hào)：1828201