【作者】 郝俊青；

【导师】 董亮；

【作者基本信息】 山东大学 ， 内科学， 2010， 硕士

【摘要】 研究目的：支气管哮喘(简称哮喘)是一种由多种炎症细胞和炎症因子参与的慢性气道炎症性疾病,“卫生假说”并不能解释所有的哮喘现象。哮喘病因学研究显示,哮喘是遗传和环境因素作用的结果,或者说是固有免疫系统和获得性免疫系统相互作用的结果。所以,研究哮喘病因学中两个免疫系统间的相互作用,将会有助于进一步明确哮喘的病因和发病机制。近年来,室内灰尘、内毒素、病毒感染等在哮喘气道炎症中的作用越来越受到关注,室内灰尘(HDM)是存在于周围环境中的一类重要的病原微生物成分,我们试图运用HDM干预小鼠哮喘模型,研究HDM通过TLR4信号转导途径诱导肺泡巨噬细胞(AM)活化,探讨TLR4激活进而诱导AM的活化在哮喘小鼠气道炎症中的作用,从而更加深入明确哮喘的病因和发病机制。研究方法：雌性BALB/c小鼠30只,随机分为哮喘模型组(OVA) A, HDM处理组(100mg/L HDM+OVA) B和对照组(生理盐水)C。用卵白蛋白(OVA)致敏与激发建立小鼠哮喘模型；苏木素伊红(HE)染色观察小鼠气道及肺组织病理变化；光学显微镜下观察小鼠支气管肺泡灌洗液(BALF)中细胞分类及计数；酶联免疫吸附试验(ELISA)检测各组小鼠支气管肺泡灌洗液(BALF)上清中IL-4、IL-5、IL-13和IFN-γ的含量；实时定量聚合酶链反应(RT-PCR)法测定AM的TLR4的表达,流式细胞技术(FCM)检测AM的CD80、CD86的表达。统计学分析：检测结果均以均数±标准差(x-±s)表示,采用SPSS13.0统计软件处理,各组间比较采用方差分析,组间两两比较采用LSD法,P<0.05为差异有统计学意义。结果：1.小鼠哮喘模型成功建立的临床表现以小鼠出现明显的烦躁不安,活动频繁,呼吸急促,腹肌抽搐,大、小便失禁,毛发竖起,部分小鼠出现反应迟钝,动作迟缓等为哮喘成功的标志。2.HE染色观察小鼠肺组织病理学改变。高倍显微镜下观察可见,A组支气管黏膜下水肿,黏液腺增生,黏膜及黏膜下层、平滑肌外均可见大量以嗜酸性粒细胞为主的炎症细胞浸润；B组肺泡隔断裂,肺间质充血。肺泡腔内中性粒细胞和淋巴细胞大量浸润；C组气道上皮无增厚,气道周围无炎症细胞浸润,肺泡壁结构完整。3.各组小鼠BALF中炎症细胞的表达水平与A组相比,B组细胞总数、中性粒细胞水平显著增高,差异有统计学意义(P<0.05),而嗜酸性粒细胞差异无统计学意义(P>0.05)。4.各组小鼠BALF上清中IL-4、IL-5、IL-13和IFN-γ的表达水平A组BALF上清中IL-4、IL-5、IL-13和IFN-γ水平显著高于C组,差异有统计学意义(P<0.05),B组BALF上清中IL-4、IL-5和IL-13的水平显著高于A组(P<0.05),而IFN-γ差异无统计学意义(P>0.05)。5.各组小鼠AM的TLR4的表达水平与A组相比,B组AM的TLR4mRNA的表达能力显著增强,差异有统计学意义(P<0.05),而A组与C组AM的TLR4mRNA表达能力差异无统计学意义(P>0.05)。6.各组小鼠AM中CD80、CD86的表达水平与A组相比,B组CD80分子表达差异无统计学意义(P>0.05),CD86表达能力显著增强(P<0.05)。结论：1.AM的TLR4是介导HDM引起免疫应答和机体炎症的主要受体,HDM可诱导哮喘小鼠AM的TLR4mRNA的表达上调,而OVA并不能刺激TLR4表达增强。2.CD86协同Th2的产生,促进体液免疫,下调细胞免疫,Th2细胞克隆的产生及炎症介质的释放更加依赖于CD86/CTLA4相互作用所提供的共刺激信号。3.HDM通过AM的TLR4的高表达诱导AM的活化,加重哮喘的气道炎症。意义：我们通过HDM干预小鼠哮喘模型,进一步明确了HDM通过TLR4信号转导途径诱导AM活化,以及TLR4激活进而诱导AM的活化在哮喘小鼠气道炎症中的作用,从而更加深入明确了哮喘的病因和发病机制。更多还原

【Abstract】 Objectives:Bronchial asthma is a disease characterized by chronic airway inflammation, many inflammatory cells and inflammatory factors contributed to its development. "Hygiene hypothesis" can only partially explain the increasing incidence rate of asthma. Asthma is the result of both heredity and environment (in other words, inherent immunity and acquired immunity). Research on the interactions between these two immune systems will provide more conclusive information on the etiology and mechanism of asthma.Nowadays, researchers are paying more and more attention on the contribution of house dust mite, endotoxin, viral infection to asthma. House dust mite(HDM) is an important part of pathogen in the surrounding environment, we treat the asthma mouse model with the HDM. We intend to investigate the activation of TLR4 by HDM, and then the activation of AM by TLR4. We aim at confirming the airway inflammation caused by AM activation, so as to identify the etiology and mechanism of asthma.Methods:30 female BALB/c mice were randomly divided into OVA group (A), HDM+OVA group (B) and control group (C). We used OVA sensitization and challenge to induce a murine asthmatic model. HE stain was used to monitor the pathological changes of airway and lung. The optical microscope was used to observe the cells from BALF; The concentration of IL-4、L-5、L-13 and IFN-γwas determined by ELISA; The expression of AM and TLR4 was measured by Real-time PCR; The expression of CD80 and CD86 was detected by FCM.Results:1. In group A, bronchus sub-mucosa edema, mucus gland hyperplasia, eosinophile granulocyte dominated inflammatory cells infiltration among all layers; In group B, breakage of interalveolar septum, lung interstitium congestion, neutrophile granulocyte and leukomonocyte dominated inflammatory cells infiltration in the alveolar space; In group C, no airway endepidermis hyperplasia, no inflammatory cells infiltration, intact alveolar wall.2. The total cell count, neutrophile granulocyte count of group B was significantly higher than group A, but the eosinophile granulocyte count was similar.3. The concentration of IL-4, IL-5, IL-13 and IFN-y in BALF of group A was significantly higher than group C, the concentration of IL-4, IL-5 and IL-13 in BALF of group B was significantly higher than group A, the concentration of IFN-γwas similar.4. The expression of AM and TLR4 mRNA in group B was statistically higher than group A, while group A and C was similar.5. The expression of CD86 of group B was significantly higher than Group A, the CD80 expression was similar.Conclusions:HDM will activate the AM through TLR4 passway so as to accelerate the airway inflammation.更多还原