本文選題：哮喘 + GABAAαR��； 參考：《鄭州大學(xué)》2012年碩士論文

【摘要】：背景及目的 支氣管哮喘(bronchial asthma)是由多種細(xì)胞和細(xì)胞組分參與的氣道慢性炎癥性疾病,粘液過(guò)度分泌是重度哮喘的特征之一,粘液阻塞氣道是哮喘致死的重要原因。由于空氣污染、吸煙等原因,近年來(lái)支氣管哮喘的發(fā)病率逐漸增加,盡管支氣管哮喘的免疫學(xué)機(jī)制和氣道炎癥學(xué)說(shuō)已被大家認(rèn)同,但仍不能完全解釋支氣管哮喘的所有病理生理變化。神經(jīng)、內(nèi)分泌和免疫系統(tǒng)之間著廣泛而密切的聯(lián)系,形成神經(jīng)內(nèi)分泌免疫學(xué),又稱(chēng)神經(jīng)免疫調(diào)節(jié),在支氣管哮喘的發(fā)病機(jī)制中發(fā)揮著重要作用。神經(jīng)系統(tǒng)可以通過(guò)外周神經(jīng)纖維、神經(jīng)遞質(zhì)、神經(jīng)內(nèi)分泌激素或神經(jīng)生長(zhǎng)因子等影響免疫系統(tǒng)的功能。下丘腦(hypothalamus)是神經(jīng)免疫調(diào)節(jié)的重要中樞,近來(lái)研究證明哮喘發(fā)作與下丘腦等腦區(qū)活動(dòng)及腦內(nèi)遞質(zhì)含量密切相關(guān),外源性增加腦內(nèi)某些遞質(zhì)如組胺的含量,或阻斷某些遞質(zhì)作用通路,可影響氣道炎癥和引起氣道高反應(yīng)性,進(jìn)而導(dǎo)致支氣管哮喘的發(fā)生；哮喘發(fā)作時(shí),下丘腦室旁核(PVN)內(nèi)Fos蛋白表達(dá)顯著增加,這些研究說(shuō)明支氣管哮喘與神經(jīng)免疫調(diào)節(jié)密切相關(guān)。Y-氨基丁酸(y-aminobutyric acid, GAB A)是哺乳動(dòng)物重要的抑制性神經(jīng)遞質(zhì)之一,廣泛分布于中樞神經(jīng)系統(tǒng)。研究發(fā)現(xiàn)實(shí)驗(yàn)哮喘小鼠或哮喘患者肺上皮細(xì)胞表達(dá)GABAAR及GABA合成酶谷氨酸脫羧酶65/67(GAD65/67)增多,但小鼠腦組織中GABAAR的變化尚不清楚。本實(shí)驗(yàn)通過(guò)建立對(duì)照組、哮喘組和地塞米松干預(yù)組小鼠模型,檢測(cè)各組小鼠肺和下丘腦中γ-氨基丁酸A受體α亞基(GABAARα)、肺組織黏蛋白基因MUC5AC和肺泡灌洗液(BALF)中白介素13(IL-13)表達(dá)量的變化,分析哮喘組小鼠肺部GABAARα、IL-13與MUC5AC、下丘腦GABAARa與肺部GABAARa變化的關(guān)系,以及地塞米松的干預(yù)作用,為深入了解哮喘的發(fā)病機(jī)制提供實(shí)驗(yàn)依據(jù)。 材料方法 6周齡清潔級(jí)雌性BALB/C小鼠30只,按隨機(jī)數(shù)字表法分為3組,即對(duì)照組、哮喘組、地塞米松組,每組10只。第1d和第8d腹腔注射0.1%OVA/AL(OH)3混懸液1ml致敏,第15d開(kāi)始以1%OVA霧化激發(fā)30min,連續(xù)7d,對(duì)照組致敏與激發(fā)均以生理鹽水代替,地塞米松組在激發(fā)前腹腔注射地塞米松1mg/Kg。最后一次激發(fā)后用10%水合氯醛(20mg/kg)腹腔注射麻醉小鼠,暴露胸腔,氣管插管取肺肺泡灌洗液(BALF);切取左肺，，上葉“用10%多聚甲醛固定,常規(guī)石蠟包埋切片備用；左肺下葉迅速放入液氮中,速凍后轉(zhuǎn)移至-80℃冰箱備用；打開(kāi)顱腔,在顯微鏡下迅速分離出下丘腦,速凍后放入-80℃冰箱備用。肺泡灌洗液離心,行細(xì)胞計(jì)數(shù)和分類(lèi)計(jì)數(shù)；用ELISA(酶聯(lián)免疫吸附測(cè)定)法檢測(cè)BALF中的IL-13的含量；肺組織切片行HE染色；用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)法(RT-PCR)和免疫組化法檢測(cè)肺組織GABAARa的表達(dá)；用RT-PCR檢測(cè)肺組織中MUC5ACmRNA和下丘腦中GABAARa的表達(dá)水平。實(shí)驗(yàn)數(shù)據(jù)用SPSS17.0進(jìn)行統(tǒng)計(jì)分析。 結(jié)果 1小鼠一般情況：OVA激發(fā)后哮喘組小鼠出現(xiàn)典型的哮喘癥狀如活動(dòng)少、打噴嚏、搔鼻、點(diǎn)頭樣喘息；地塞米松組上述癥狀較哮喘組稍輕；對(duì)照組無(wú)喘息癥狀。 2BALF細(xì)胞計(jì)數(shù)及分類(lèi)計(jì)數(shù)和IL-13的濃度：哮喘組小鼠BALF中細(xì)胞以單核細(xì)胞和嗜酸性粒細(xì)胞為主,其細(xì)胞總數(shù)和中性粒細(xì)胞、嗜酸性粒細(xì)胞、淋巴細(xì)胞比率均較對(duì)照組明顯增多(P0.05),地塞米松治療組與哮喘組相比細(xì)胞總數(shù)及中性粒細(xì)胞、嗜酸性粒細(xì)胞、淋巴細(xì)胞比率均減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。哮喘組BALF中IL-13濃度為(47.17±0.70)pg/ml,地塞米松組為(24.48±0.60)pg/ml,對(duì)照組為(11.15±0.55)pg/ml,各組差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 3肺組織HE染色：哮喘組小鼠支氣管壁及血管壁周?chē)写罅垦装Y細(xì)胞浸潤(rùn),氣道上皮不完整,肺泡間隔增寬,黏膜層增厚并充血水腫,肺泡腔內(nèi)有炎性滲出物；地塞米松組炎癥細(xì)胞較哮喘組減少；對(duì)照組小鼠肺泡壁結(jié)構(gòu)完整,氣道黏膜正常,氣道周?chē)鸁o(wú)炎癥細(xì)胞浸潤(rùn)。 4肺組織中GABAARα mRNA、蛋白和MUC5AC mRNA表達(dá)情況：肺部GABAARα免疫組化陽(yáng)性表達(dá)物主要表達(dá)于氣道上皮細(xì)胞,在基底部無(wú)表達(dá)。哮喘組GABAARα mRNA、蛋白和MUC5AC mRNA表達(dá)均較對(duì)照組和地塞米松干預(yù)組增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。經(jīng)過(guò)地塞米松治療,肺部GABAARα和MUC5AC較哮喘組明顯降低,但仍較正常組高(P0.05)。 5下丘腦GABAARα mRNA表達(dá)情況：哮喘組下丘腦GABAARα mRNA表達(dá)量較對(duì)照組增高,地塞米松干預(yù)組表達(dá)量較哮喘組減少,各組差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。 6哮喘組小鼠各指標(biāo)相關(guān)性分析：哮喘組小鼠肺組織GABAARα mRNA表達(dá)與MUC5AC mRNA表達(dá)呈正相關(guān)(r=0.909,P0.05)；BALF中IL-13與肺組織MUC5AC mRNA表達(dá)呈正相關(guān)(r=0.925,P0.05)；下丘腦與肺組織中GABAARα mRNA表達(dá)呈正相關(guān)(r=0.727,P0.05)。 結(jié)論 1哮喘小鼠肺和下丘腦GABAARα表達(dá)增加,提示GABAARα與哮喘發(fā)病密切相關(guān),GABA系統(tǒng)可能與哮喘神經(jīng)免疫調(diào)節(jié)有關(guān)。 2GABAARα可能參與介導(dǎo)炎癥因子IL-13引起的氣道粘液過(guò)度分泌。 3地塞米松可以抑制下丘腦和肺組織中GABAARα的含量,降低MUC5AC的表達(dá),對(duì)哮喘起治療作用。
[Abstract]:Background and Purpose

Bronchial asthma ( bronchial asthma ) is a chronic inflammatory disease caused by various cellular and cellular components , and mucus hypersecretion is one of the most important causes of asthma .
The expression of GABA偽 , IL - 13 and glutamic acid decarboxylase 65 / 67 ( GAD65 / 67 ) in the lung epithelial cells of asthmatic mice were investigated .

Material method

In 6 - week - old female BALB / C mice , 30 male BALB / C mice were randomly divided into three groups : control group , asthma group and dexamethasone group . Results In mice with asthma after OVA challenge , the typical symptoms of asthma in the asthmatic group were less than those in the asthma group ; the above - mentioned symptoms in the dexamethasone group were slightly lighter than those in the asthma group ; the control group had no asthmatic symptoms . The cell count of BALF and the concentration of IL - 13 in asthmatic group were significantly higher than those in the control group ( P0.05 ) . The concentrations of IL - 13 in BALF were ( 47.17 鹵 0.70 ) pg / ml , ( 24.48 鹵 0.60 ) pg / ml , and ( 11.15 鹵 0.55 ) pg / ml in the control group ( P0.05 ) . 3 lung tissue HE staining : there were a large number of inflammatory cell infiltration around the bronchial wall and the vessel wall of the asthmatic group , the airway epithelium was incomplete , the alveolar septum was widened , the mucosal layer thickened and the congestion was edema , inflammatory exudate was found in the alveolar cavity ; the inflammatory cells in the dexamethasone group were smaller than those in the asthma group ; the alveolar wall of the control group was intact , the airway mucosa was normal , and there was no infiltration of inflammatory cells around the airways . The expression of GABA偽 mRNA , protein and MUC5AC mRNA in lung tissues was significantly higher than that in control group and dexamethasone group ( P0.05 ) . The expression of GABA偽 mRNA in the hypothalamus of the hypothalamus was higher than that in the control group . The expression of GABA偽 mRNA in the hypothalamus of the asthmatic group was higher than that in the control group , and the expression level of the dexamethasone intervention group was lower than that in the asthma group ( P0.05 ) . There was positive correlation between the expression of GABA偽 mRNA and MUC5AC mRNA in lung tissues of asthmatic group ( r = 0.909 , P0.05 ) . The expression of GABA偽 mRNA in BALF was positively correlated ( r = 0.727 , P0.05 ) . Conclusion The expression of GABA偽 in the lung and hypothalamus of asthmatic mice increased , suggesting that GABA偽 is closely related to the pathogenesis of asthma , and the GABA system may be related to the immune regulation of asthma . 2GABARRA偽 may be involved in mediating hypersecretion of airway mucus caused by inflammatory cytokines IL - 13 . Dexamethasone could inhibit the content of GABA偽 in the hypothalamus and lung tissues , reduce the expression of MUC5AC , and play a role in the treatment of asthma .
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R562.25

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