【作者】 王苹莉；

【导师】 沈华浩；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 支气管哮喘(简称哮喘)是一种以嗜酸性粒细胞(Eosinophils, Eos)、肥大细胞和T淋巴细胞浸润为主的气道慢性炎症性疾病,表现为气道高反应性和可逆性气流受限。气道炎症的反复发生或持续存在会使气道出现不可逆的结构改变,称为气道重塑,其特征主要有：气道周围平滑肌增生／肥大,上皮下纤维化和胶原沉积,上皮细胞黏液化生和黏液高分泌以及微血管的增生。当哮喘发展至气道重塑阶段会出现气流受限的不完全可逆,患者对抗炎治疗的反应下降而使预后变差。哮喘严重威胁人类健康,其发病率在全世界范围内有增高趋势。目前全球有超过3亿哮喘患者,我国约有3900万哮喘患者。现行的以吸入糖皮质激素为首选的治疗方案能控制哮喘症状,但需长期用药,不能预防及根治其发生,也不能抑制气道重塑的发生。如何从哮喘的发病机制入手寻找一有效的哮喘预防手段是目前面临的难题。现有研究表明哮喘是一种免疫失衡性疾病,哮喘的发生与机体IFN-γ的微环境缺失和Th2细胞免疫的过度存在相关,大量Th2细胞因子(IL-4, IL-5, IL-13等)的增加,促进了IgE的释放,致使肺组织嗜酸性粒细胞浸润和气道黏液高分泌,导致气道反应性的增高和气道重塑的发生。通过纠正机体失衡的Th1/Th2免疫反应成为预防哮喘研究的热点和靶点。减毒活菌卡介苗(Bacillus Calmette-Guerin, BCG)是一种很强的Thl免疫刺激剂,多项动物研究显示,BCG干预可明显抑制抗原致敏激发所致的气道Eos浸润,降低气道高反应性。流行病学研究关于人体BCG接种与哮喘发生的关系虽然有争议,但是最近一项对23项临床研究的荟萃分析结果表明,人类生命早期接种BCG疫苗能够降低儿童哮喘发生率,进一步支持了BCG接种能够预防哮喘发生的观点。BCG接种对哮喘的抑制作用与多种因素有关,包括BCG结构重组,采用不同的接种途径、接种次数以及改变接种时间窗等。多年来我们实验室致力于寻求一种最有效的、安全的、有望应用于人类的接种方式。通过一系列的前期研究,我们发现新生儿期幼鼠多次皮下给予小剂量BCG接种可以抑制成年后哮喘的发生,并且该保护作用能维持至老年。既然BCG早期多次接种能长期抑制哮喘气道炎症,那么是否可以进一步抑制与炎症持续存在相关的气道重塑的发生呢?目前为止,有关早期多次BCG接种对哮喘气道重塑的影响尚未见报道。其次是关于BCG接种抑制哮喘发生的免疫机制问题。虽然多数研究均证实BCG是一很强的Thl免疫刺激剂,但随着调节性T细胞(regulatory T cells, Treg)和新型Th细胞(Thl7细胞)的发现以及功能的不断研究,BCG是否通过Treg或Th17来抑制哮喘发生也受到重视。有研究发现IL-17能够促进机体对结核杆菌最佳的Thl免疫反应,介导T细胞分泌IFN-γ,并促进表达IFN-γ的CD4+T细胞向肺内迁移,从而在结核杆菌疫苗干预哮喘的过程中发挥作用。也有研究认为结核杆菌疫苗能够通过诱导Treg增殖抑制哮喘炎症。但以上研究多基于急性炎症模型或体外研究。而现有研究发现,慢性或重症哮喘与急性或轻症哮喘患者中多种炎症因子的表达存在明显差异,如在慢性持续性或重症哮喘患者中Th17细胞比例和IL-17的表达明显高于轻症患者。基于本研究中我们使用的是慢性气道重塑模型,我们有兴趣观察BCG早期接种在慢性哮喘中的免疫调节模式,而这方面目前鲜见报道。由此,本实验分两部分进行研究：(1)通过构建小鼠哮喘气道重塑模型,观察新生期多次BCG接种对小鼠哮喘气道重塑发生的影响；(2)通过流式细胞术和real-time PCR检测早期多次BCG接种后肺组织T细胞亚群的变化,从而了解早期多次皮下接种BCG在气道重塑模型中的免疫应答情况。第一部分小鼠哮喘气道重塑模型的建立及早期多次卡介苗接种对气道重塑的影响目的：通过抗原致敏和反复激发建立具有典型气道重塑特征的慢性哮喘模型。观察早期多次卡介苗接种对哮喘气道重塑的影响。方法：采用BALB/c新生鼠,分为OVA致敏激发组(OVA组)、早期多次BCG接种组(BCG+OVA组)和生理盐水阴性对照组(SHAM组)。BCG+OVA组小鼠分别在出生0、7、14天予以BCG105CFUs的剂量行皮下接种3次,OVA组和SHAM组注射等体积注射用水。小鼠长大至5周时予OVA腹腔注射致敏,2周后第二次致敏,10天后以1%OVA雾化吸入激发,每次40min,每周连续雾化吸入3天,连续8周。最后一次激发后24小时行气道反应性测定。48小时收集标本,行支气管肺泡灌洗液细胞分类计数,肺组织病理切片观察气道炎症、气道周围平滑肌增生、胶原沉积和黏液分泌情况。结果：OVA组小鼠经抗原致敏和反复激发后出现了典型的气道重塑特征,包括气道周围平滑肌明显增生、胶原沉积增加、气道黏液高分泌以及气道和血管周围大量炎症细胞浸润,同时气道反应性明显增高。而早期多次BCG接种后的小鼠(BCG+OVA组)BALF中炎症细胞总数减少,尤其Eos减少更明显(P<0.01)。气道周围平滑肌增生和胶原沉积较OVA组减少,尤其以直径150-250μm的小气道周围减少明显(P<0.05)。气道反应性也较OVA组降低(P<0.05)。结论：我们通过OVA抗原致敏和反复雾化吸入激发的方法构建了稳定的具有典型气道重塑特征和气道高反应性的小鼠慢性哮喘模型。新生鼠早期多次BCG接种可降低慢性哮喘模型气道周围炎症细胞尤其是Eos的浸润,减轻气道周围平滑肌增生和胶原沉积,降低气道高反应性,其中对小气道周围气道重塑的抑制较中大气道更为明显。第二部分早期多次卡介苗接种减轻哮喘气道重塑的相关免疫机制目的：观察早期多次BCG接种后肺组织T细胞免疫应答的变化。观察BCG接种对肺组织中Eos活化因子和趋化因子表达的影响,以及对影响气道重建的重要因子TGF-β1表达的影响。方法：最后一次抗原激发后48小时行肺泡灌洗,ELISA法检测灌洗液上清中IFN-γ、IL-5、IL-17、IL-13、eotaxin-1、TGF-β1的水平。分离肺组织淋巴细胞,经PMA刺激后行表面抗体标记(CD3, CD4, CD8, CD25),细胞破膜处理后标记胞内抗体(IFN-γ, IL-4, IL-17A, IL-10, Foxp3),流式细胞术检测和分析各T’细胞亚群的比例。另取肺组织提取RNA,经逆转录后用real-time PCR法检测IFN-γ, IL-17A和Fopx3mRNA的表达。肺组织石蜡切片行TGF-β1免疫组化染色,图像分析计数气道基底膜外围30μm范围内TGF-β1+细胞数。结果：与SHAM组相比,OVA组表达IL-4和表达IL-17的T细胞比例增高,而表达IFN-γ的T细胞比例无明显变化。BCG多次接种后肺组织中表达IFN-γ的T细胞明显增高达4倍,尤其表现为Thl细胞的增加(5.01±0.66%vs 0.83±0.18%ofCD3+T cells, BCG+OVA vs OVA, P<0.01). Thl7细胞、Th2细胞和Treg在OVA组和BCG+OVA组之间无明显差别。real-time PCR检测结果与流式分析的结果一致,BCG接种后仅发现促进IFN-γmRNA明显增高,而IL-17A和Fopx3 mRNA水平在BCG+OVA组与OVA组之间无明显差异,但这两组的IL-17A mRNA水平均较SHAM组明显增高。用ELISA方法检测BALF中细胞因子表达水平,结果显示：OVA组和SHAM组EFN-γ水平无明显差异,BCG接种组IFN-γ较未接种组明显增高(P<0.05)。OVA组BALF中IL-5和Eotaxin-1水平较SHAM组显著增加,BCG+OVA组IL-5和Eotaxin-1水平均下降,两组间相比差异有显著性。IL-13水平OVA组较SHAM组高,BCG+OVA组较OVA组有下降,但统计无显著差异。IL-17水平各组中均很低,在检测的敏感性之外。BALF中TGF-β1水平用ELISA方法检测结果显示：OVA组BALF中TGF-β1水平较SHAM组显著增加,BCG+OVA组TGF-β1水平较OVA组下降,两组间相比差异有显著性。肺组织切片TGF-β1免疫组化染色发现：OVA组气道周围TGF-β1染色阳性的细胞数较SHAM组明显增高,BCG接种组较OVA组降低。结论：早期多次BCG接种导致肺组织中Thl细胞比例和IFN-γ水平明显升高,而对Treg无明显影响,说明早期BCG皮下接种对慢性哮喘的抑制主要通过诱导Th1免疫而非通过Treg增加而起作用。Th17细胞和IL-17参与了慢性哮喘炎症和气道重塑发生的过程,但早期皮下接种BCG并不影响其表达。早期多次BCG接种降低了肺组织中IL-5和TGF-β1的水平,可能与气道重塑的减轻相关。总结我们实验室前期研究发现,早期多次BCG接种可降低哮喘急性模型的气道炎症,且该作用可持续至老年。本研究在此基础上,进一步观察了早期多次BCG皮下接种对小鼠慢性哮喘气道重塑模型的影响及肺组织T细胞免疫应答的变化,得出如下结论：1、通过OVA抗原致敏和反复雾化吸入激发,成功建立了稳定的具有典型气道重塑特征和气道高反应性的小鼠慢性哮喘模型。2、早期多次BCG接种可减轻小鼠慢性哮喘模型的气道周围平滑肌增生和胶原沉积,降低气道高反应性,减轻气道炎症细胞浸润,尤其Eos浸润明显抑制。3、早期多次BCG接种降低了肺组织中IL-5和TGF-β1的水平,可能与气道重塑的减轻相关。4、早期多次BCG皮下接种导致肺组织中Th1细胞比例和IFN-γ水平明显升高,而对Treg无明显影响,说明早期BCG皮下接种对慢性哮喘的抑制主要通过诱导Thl免疫而非通过Treg增加而起作用。5、Th17细胞和IL-17参与了慢性哮喘炎症和气道重塑发生的过程,但早期皮下接种BCG并不影响其表达。更多还原

【Abstract】 Asthma is a chronic airway inflammatory disease, which is characterized by the presence of increased numbers of eosinophils and a predominantly Th2 response. A persistent Th2 response leads to airway remodeling, characterized by increased globet cell numbers and consequent mucus production, smooth muscle hypertrophy and hyperplasia, collagen and elastic fiber deposition, and increased angiogenesis.Current asthma management strategies principally involve the use of inhaled corticosteroids to control symptoms, but it has little effect on inhibiting the disease progression and the development of airway remodeling. Therefore, preventative interventions may be a better approach to protect individuals from asthma. BCG as a strong inducer for Thl-type immune response, is considered as a potential candidate. Lately a mata-analysis for 23 studies suggested a protective effect of BCG exposure on childhood asthma occurrence, supports the hypothesis that exposure to the BCG vaccine in early life prevents asthma. Several factors may affect the efficacy of BCG vaccination, including the route and time of delivery. Our group has demonstrated previously that neonatal BCG vaccination has a long-term effect on inhibiting AHR and eosinophilia in mouse asthma. However, the potential of neonatal multiple BCG vaccination in preventing the development of airway remodeling associated with repeated allergen exposure remains to be addressed.Concerning the underlying mechanisms of BCG vaccination against asthma, most previous studies demonstrated that BCG inhibited Th2 response by inducing a strong Thl-type immune response. But lately some studies also suggested that BCG or killed Mycobacterium vaccae confer protection against airway inflammation by giving rise to allergen-specific regulatory T cells. Th17 is the newest member of the Th cell family. It’s function in asthma, especially in chronic and severe asthma, is receiving attention. How neonatal BCG vaccination eliciting an immunological response in an airway remodeling model remains to be addressed.In this study, using a chronic asthma model of OVA-sensitized and repeated challenged mice, we further determined if neonatal multiple BCG vaccination is capable of reducing the airway remodeling. Then we used Flow Cytometric and Real-time PCR analysis to examine the differentiation of pulmonary T cell populations and to determine the underlying mechanisms of neonatal BCG vaccination in inhibiting asthma.Part I The establishment of murine model of airway remodeling and the effect of neonatal multiple BCG vaccination on the airway remodeling of asthmaObjective:To investigate whether neonatal multiple vaccination with BCG could decrease the airway remodeling of asthma.Methods:BALB/c mice were injected intradermally with 25μl Freeze-dried living BCG suspension [105 infection units (IU)] at birth, and boosted two times on days 7,14 with the same dose of BCG. At 5 weeks of age, mice were immunized i.p. on days 0,14 with 20μg of OVA emulsified in 100μl Imject Alum. Groups of mice that had been sensitized with OVA were challenged with an aerosolized OVA thrice a week for 8 weeks. Age-and sex-matched mice that had been sensitized and challenged with saline at each time point were used as negative controls.24h after the final OVA challenge, airway hyperresponsiveness (AHR) to inhaled methacholine was measured. Mice were sacrificed 48h after the final OVA challenge and bronchoalveolar lavage fluid (BALF) and lungs were analyzed. Lungs were processed for either histologic staining (HE, PAS and Masson trichrome) or immunostaining (a-SMA). Lung inflammation, smooth muscle layer thickness, peribronchial fibrosis, airway mucus expression were analyzed.Results:OVA sensitive and repeated challenge induced airway remodeling including increased mucus production, smooth muscle hyperplasia, increased collagen deposition and airway hyperresponsiveness. Multiple BCG vaccination decreased levels of BALF total cells (30.27±3.06×104 vs 41.72±3.11×104 BCG+OVA vs OVA; p<0.05) and eosinophils (1.22±0.28×104 vs 8.46±1.34×104; BCG+OVA vs OVA; p<0.01). BCG vaccination also inhibited airway responsiveness, reduced the levels of peribronchial a-smooth muscle actin stained area (0.93±0.08 vs 1.45±0.12μm2/μm; BCG+OVA vs OVA; p< 0.05) and peribronchial collagen deposition (0.64±0.06 vs 0.88±0.09μm2/μm; BCG+OVA vs OVA;p<0.05). The mucus overproduction also decreased.Conclusion:Allergen sensitive and repeated aerosolized allergen challenge established murine model of airway remodeling. Multiple neonatal BCG vaccination reduced levels of allergen-induced airway remodeling and airway hyperresponsiveness in mice.Part II The possible mechanisms of BCG vaccination inhibiting the airway remodeling of miceObjective:To investigate the pulmonary T cell response following neonatal BCG vaccination and the possible mechanisms of BCG vaccination inhibiting the airway remodeling.Methods:48h after the final OVA challenge, mice were sacrificed and BALF was collected. Levels of selected cytokines and chemokines in BALF (IFN-y, IL-5, IL-17, IL-13, eotaxin-1, TGF-β1) were assayed using ELISA kits. Lung lymphocytes were isolated and labeled with T cells surface Abs (CD3, CD4, CD8 and CD25) and intracellular Abs (IFN-γ, IL-4, IL-17A, IL-10, Foxp3). Data were collected and analyzed using a FACScan for three-color flow cytometry. Lung tissue RNA was extracted with Trizol. Real-time PCR quantitative analyses were performed to detect the level of IFN-y, IL-17A, Foxp3mRNA. Lung tissues paraffin were also immunostained with TGF-β1 Ab, and peribronchial TGF-β1+cells were counted.Results:Neonatal BCG vaccination induced significantly increased level of BALF IFN-y and decreased the levels of BALF IL-5 and BALF eotaxin-1 compared with non-vaccinted and chronic OVA challenge alone. The level of IL-13 also decreased but had not statistical significance. Flow cytometric detection of T cell subpopulations in the lungs showed that BCG vaccination increased almost 6-fold CD3+CD8- IFN-γ+T cells (5.01±0.66 vs 0.83±0.18% of CD3+T cells) in the lungs as compared with the non-vaccinated mice. No difference was observed in the frequency of CD3+CD8- IL-4+T cells, CD3+CD8- IL-17+T cells and CD4+Foxp3+T cells(Treg) in the lungs of BCG vaccinated and non-BCG vaccinated mice, but IL-4+T cells and IL-17+T cells of OVA and BCG+OVA group mice significantly increased comparing with control mice. The results of IFN-y, IL-17A, Foxp3mRNA level in lung tissue were similar with flow cytometric detection of T cell subpopulations. Exposure of mice to chronic OVA challenge induced a significant increase in the level of TGF-β1 in BALF and peribronchial TGF-β1+cells. Multiple BCG vaccination decreased the level of TGF-β1 in BALF and peribronchial TGF-β1+cells.Conclusion:Our data suggested that neonatal multiple BCG vaccination primarily increased the level of IFN-y and IFN-y producing T cells in lungs. But has little effect on IL-17 producing T cells and pulmonary regulatory T cells. BCG vaccination decreased the level of IL-5 and Eotaxin-1 in BALF, also reduced levels of TGF-β1 in BALF and peribronchial TGF-β1+cells which may contribute to the abatement of airway remodeling. Our previous study showed that compared to other studies with single dose of BCG immunization, neonatal multiple BCG vaccination elicited longer-term protection by inhibiting allergic airway inflammation. In the present study, we further investigated the effect of multiple vaccination with BCG at newborn on the airway remodeling of chronic asthma and determined the possible immunological mechanisms. Conclusions obtained from our data were listed as following:1. We established a stable murine model of chronic asthma with typical airway remodeling features.2. Multiple neonatal BCG vaccination reduced levels of allergen-induced airway remodeling and airway hyperresponsiveness, as well as the airway inflammation.3. Multiple neonatal BCG vaccination decreased the levels of IL-5 and TGF-β1 in lungs, which may contribute to the inhibition of airway remodeling.4. Multiple BCG vaccination increased the frequency of Thl cells and IFN-y level, but not Treg cells, which suggested that the inhibition of asthma by BCG vaccination was not associated with an increased number of pulmonary regulatory T cells, but was positively correlated with the increase of IFN-y producing T cells.5. Thl 7 cells and IL-17 contribute to the development of airway remodeling. Neonatal BCG vaccination did not change the frequency of Th17 cells and IL-17 level.更多还原