实验哮喘小鼠肺和下丘脑中GABAARα的变化及其与气道黏液分泌的关系

发布时间：2018-06-08 07:17

本文选题：哮喘 + GABAAαR　；参考：《郑州大学》2012年硕士论文

【摘要】：背景及目的支气管哮喘(bronchial asthma)是由多种细胞和细胞组分参与的气道慢性炎症性疾病,粘液过度分泌是重度哮喘的特征之一,粘液阻塞气道是哮喘致死的重要原因。由于空气污染、吸烟等原因,近年来支气管哮喘的发病率逐渐增加,尽管支气管哮喘的免疫学机制和气道炎症学说已被大家认同,但仍不能完全解释支气管哮喘的所有病理生理变化。神经、内分泌和免疫系统之间着广泛而密切的联系,形成神经内分泌免疫学,又称神经免疫调节,在支气管哮喘的发病机制中发挥着重要作用。神经系统可以通过外周神经纤维、神经递质、神经内分泌激素或神经生长因子等影响免疫系统的功能。下丘脑(hypothalamus)是神经免疫调节的重要中枢,近来研究证明哮喘发作与下丘脑等脑区活动及脑内递质含量密切相关,外源性增加脑内某些递质如组胺的含量,或阻断某些递质作用通路,可影响气道炎症和引起气道高反应性,进而导致支气管哮喘的发生；哮喘发作时,下丘脑室旁核(PVN)内Fos蛋白表达显著增加,这些研究说明支气管哮喘与神经免疫调节密切相关。Y-氨基丁酸(y-aminobutyric acid, GAB A)是哺乳动物重要的抑制性神经递质之一,广泛分布于中枢神经系统。研究发现实验哮喘小鼠或哮喘患者肺上皮细胞表达GABAAR及GABA合成酶谷氨酸脱羧酶65/67(GAD65/67)增多,但小鼠脑组织中GABAAR的变化尚不清楚。本实验通过建立对照组、哮喘组和地塞米松干预组小鼠模型,检测各组小鼠肺和下丘脑中γ-氨基丁酸A受体α亚基(GABAARα)、肺组织黏蛋白基因MUC5AC和肺泡灌洗液(BALF)中白介素13(IL-13)表达量的变化,分析哮喘组小鼠肺部GABAARα、IL-13与MUC5AC、下丘脑GABAARa与肺部GABAARa变化的关系,以及地塞米松的干预作用,为深入了解哮喘的发病机制提供实验依据。材料方法 6周龄清洁级雌性BALB/C小鼠30只,按随机数字表法分为3组,即对照组、哮喘组、地塞米松组,每组10只。第1d和第8d腹腔注射0.1%OVA/AL(OH)3混悬液1ml致敏,第15d开始以1%OVA雾化激发30min,连续7d,对照组致敏与激发均以生理盐水代替,地塞米松组在激发前腹腔注射地塞米松1mg/Kg。最后一次激发后用10%水合氯醛(20mg/kg)腹腔注射麻醉小鼠,暴露胸腔,气管插管取肺肺泡灌洗液(BALF);切取左肺，，上叶“用10%多聚甲醛固定,常规石蜡包埋切片备用；左肺下叶迅速放入液氮中,速冻后转移至-80℃冰箱备用；打开颅腔,在显微镜下迅速分离出下丘脑,速冻后放入-80℃冰箱备用。肺泡灌洗液离心,行细胞计数和分类计数；用ELISA(酶联免疫吸附测定)法检测BALF中的IL-13的含量；肺组织切片行HE染色；用逆转录-聚合酶链反应法(RT-PCR)和免疫组化法检测肺组织GABAARa的表达；用RT-PCR检测肺组织中MUC5ACmRNA和下丘脑中GABAARa的表达水平。实验数据用SPSS17.0进行统计分析。结果 1小鼠一般情况：OVA激发后哮喘组小鼠出现典型的哮喘症状如活动少、打喷嚏、搔鼻、点头样喘息；地塞米松组上述症状较哮喘组稍轻；对照组无喘息症状。 2BALF细胞计数及分类计数和IL-13的浓度：哮喘组小鼠BALF中细胞以单核细胞和嗜酸性粒细胞为主,其细胞总数和中性粒细胞、嗜酸性粒细胞、淋巴细胞比率均较对照组明显增多(P0.05),地塞米松治疗组与哮喘组相比细胞总数及中性粒细胞、嗜酸性粒细胞、淋巴细胞比率均减少,差异有统计学意义(P0.05)。哮喘组BALF中IL-13浓度为(47.17±0.70)pg/ml,地塞米松组为(24.48±0.60)pg/ml,对照组为(11.15±0.55)pg/ml,各组差异具有统计学意义(P0.05)。 3肺组织HE染色：哮喘组小鼠支气管壁及血管壁周围有大量炎症细胞浸润,气道上皮不完整,肺泡间隔增宽,黏膜层增厚并充血水肿,肺泡腔内有炎性渗出物；地塞米松组炎症细胞较哮喘组减少；对照组小鼠肺泡壁结构完整,气道黏膜正常,气道周围无炎症细胞浸润。 4肺组织中GABAARα mRNA、蛋白和MUC5AC mRNA表达情况：肺部GABAARα免疫组化阳性表达物主要表达于气道上皮细胞,在基底部无表达。哮喘组GABAARα mRNA、蛋白和MUC5AC mRNA表达均较对照组和地塞米松干预组增高,差异有统计学意义(P0.05)。经过地塞米松治疗,肺部GABAARα和MUC5AC较哮喘组明显降低,但仍较正常组高(P0.05)。 5下丘脑GABAARα mRNA表达情况：哮喘组下丘脑GABAARα mRNA表达量较对照组增高,地塞米松干预组表达量较哮喘组减少,各组差异均有统计学意义(P0.05)。 6哮喘组小鼠各指标相关性分析：哮喘组小鼠肺组织GABAARα mRNA表达与MUC5AC mRNA表达呈正相关(r=0.909,P0.05)；BALF中IL-13与肺组织MUC5AC mRNA表达呈正相关(r=0.925,P0.05)；下丘脑与肺组织中GABAARα mRNA表达呈正相关(r=0.727,P0.05)。结论 1哮喘小鼠肺和下丘脑GABAARα表达增加,提示GABAARα与哮喘发病密切相关,GABA系统可能与哮喘神经免疫调节有关。 2GABAARα可能参与介导炎症因子IL-13引起的气道粘液过度分泌。 3地塞米松可以抑制下丘脑和肺组织中GABAARα的含量,降低MUC5AC的表达,对哮喘起治疗作用。
[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伪 , 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 卤 0.70 ) pg / ml , ( 24.48 卤 0.60 ) pg / ml , and ( 11.15 卤 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伪 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伪 mRNA in the hypothalamus of the hypothalamus was higher than that in the control group . The expression of GABA伪 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伪 mRNA and MUC5AC mRNA in lung tissues of asthmatic group ( r = 0.909 , P0.05 ) . The expression of GABA伪 mRNA in BALF was positively correlated ( r = 0.727 , P0.05 ) . Conclusion The expression of GABA伪 in the lung and hypothalamus of asthmatic mice increased , suggesting that GABA伪 is closely related to the pathogenesis of asthma , and the GABA system may be related to the immune regulation of asthma . 2GABARRA伪 may be involved in mediating hypersecretion of airway mucus caused by inflammatory cytokines IL - 13 . Dexamethasone could inhibit the content of GABA伪 in the hypothalamus and lung tissues , reduce the expression of MUC5AC , and play a role in the treatment of asthma .
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25

【参考文献】