肺炎链球菌7价结合疫苗免疫抑制实验性哮喘的作用及机制研究

发布时间：2018-01-25 02:08

本文关键词： 婴儿期PCV7免疫实验性哮喘成年早期 Th2 Th17 Foxp3+Treg 不成熟DCs 成年早期哮喘婴儿期PCV7免疫　出处：《重庆医科大学》2014年博士论文　论文类型：学位论文

【摘要】：背景和目的支气管哮喘(哮喘，asthma)是最常见的慢性气道炎症性疾病，其发病率呈持续上升趋势。目前临床干预哮喘的方法都只能控制哮喘症状，人们一直在努力寻找有效的哮喘防治方法。研究表明CpG-ODNs、BCG可以抑制小鼠模型的过敏性气道炎症，，然而CpG-ODN治疗可以引起有害的副作用，BCG在人类的研究中对过敏性哮喘无效。肺炎链球菌是一种常见的呼吸道病原，可以引起肺炎、中耳炎、脑膜炎和败血症。流行病学研究发现肺炎链球菌7价结合疫苗（PCV7）可以减少儿童及成人哮喘的发病率及相关住院率。也有研究表明该疫苗在成年期小鼠免疫可以通过促进Treg和抑制Th2细胞的产生抑制哮喘的特征性表现。目前PCV7用于婴儿期免疫预防儿童肺炎链球菌感染，是否婴儿期PCV7免疫可以改变成年早期实验性哮喘CD4+T细胞亚类及抑制变应性气道炎症还不清楚。树突状细胞（DCs）是专职抗原呈递细胞，能激活初始CD4+T细胞，既诱导免疫反应，又参与免疫耐受，这与DCs功能状态有关，调节DCs功能状态有助于治疗和预防哮喘。髓样DCs在IL-4、粒巨噬细胞集落刺激因子(GM-CSF)诱导下形成不成熟DCs，经抗原刺激后变为成熟DCs，表达高水平CD80、CD86、CD40等共刺激分子。我们前期研究发现疫苗BCG能诱导DCs部分成熟，部分成熟DCs促进Treg表达增加参与免疫耐受。提示DCs可能参与了PCV7抗哮喘的免疫调节。本研究旨在探讨婴儿期PCV7免疫对成年早期实验性哮喘的影响及作用机制，为哮喘防治提供新的干预思路。第一部分婴儿期PCV7免疫对成年早期实验性哮喘小鼠气道炎症、CD4+T细胞亚类的影响背景：肺炎链球菌是儿童社区获得性呼吸道感染的第一位细菌病原体，WHO推荐儿童接种PCV7预防肺炎链球菌疾病， PCV7能明显减少儿童哮喘发作次数、减轻哮喘严重程度，PCV7能预防哮喘。该疫苗在成年期小鼠免疫可以通过促进Treg的产生和抑制Th2细胞的产生抑制哮喘的特征性表现。目前PCV7用于婴儿期免疫预防儿童肺炎链球菌感染，是否婴儿期PCV7免疫可以改变成年早期CD4+T细胞亚类及抑制过敏性气道炎症还不清楚。因此本研究探讨婴儿期PCV7免疫对成年早期实验性哮喘小鼠气道炎症及CD4+T细胞亚类的影响，为后期研究奠定基础。目的：探讨婴儿期PCV7免疫对成年早期实验性哮喘小鼠气道炎症及CD4+T细胞亚类的影响。方法：清洁级3周BALB/c小鼠随机分为对照组（Control组）、PCV7免疫实验性哮喘组（PCV7+OVA组）、实验性哮喘组（OVA组），PCV7+OVA组3周时鼻腔滴注PCV7，PCV7+OVA组及OVA组用OVA（卵清蛋白）致敏与激发。小鼠肺功能仪测定小鼠气道高反应性；HE染色观察小鼠气道及肺组织病理变化；光学显微镜观察小鼠支气管肺泡灌洗液白细胞分类计数；ELISA检测BALF中INF-γ、IL-4、IL-5、IL-13、IL-17A、TGF-β及IL-10；流式细胞术检测CD4+Th细胞各亚群表达情况。结果：PCV7+OVA组小鼠哮喘症状较OVA组减轻；气道高反应性显著低于OVA组小鼠；气道炎症较OVA组明显减轻。PCV7+OVA组BALF中细胞总数、嗜酸性粒细胞、中性粒细胞较OVA组明显减少，PCV7+OVA组BALF中IL-13、IL-17A显著低于OVA组，而IFN-γ、IL-10显著高于OVA组， PCV7+OVA组纵隔淋巴结CD4+CD25+Foxp3+Treg、 CD4+INF-γ+T细胞显著高于OVA组，而CD4+IL-4+T、CD4+IL-17+T细胞显著低于OVA组。结论：婴儿期PCV7免疫是预防成年早期BALB/c小鼠实验性哮喘的有效方法。第二部分PCV7免疫对DCS影响的机制研究背景：婴儿期PCV7免疫可以促进CD4+CD25+Foxp3+Treg、Th1细胞产生，抑制Th2、Th17细胞产生，用于预防成年早期哮喘，但其机制尚不清楚。树突状细胞（DCs）是专职抗原呈递细胞，能激活初始CD4+T细胞，既诱导免疫反应，又参与免疫耐受，这与DCs功能状态有关，调节DCs功能状态有助于治疗和预防哮喘。髓样DCs在IL-4、粒巨噬细胞集落刺激因子(GM-CSF)诱导下形成不成熟DCs，经抗原刺激后变为成熟DCs，表达高水平MHCⅡ和CD80、CD86、CD40等共刺激分子。DCs摄取PCV7后如果共刺激分子的表达能够降低处于未成熟或部分成熟状态即可参与免疫耐受的调节，从而预防和治疗哮喘。本部分从体内和体外两个方面研究PCV7对DCs成熟度的影响，旨在探讨婴儿期PCV7免疫预防成年早期哮喘的可能发生机制，为哮喘防治提供新的干预思路。目的：研究体内和体外PCV7对DCs的影响，探讨PCV7预防哮喘的可能发生机制。方法：清洁级3周BALB/c小鼠分为对照组（Control组）、PCV7免疫实验性哮喘组（PCV7+OVA组）、实验性哮喘组（OVA组），PCV7+OVA组3周时鼻腔滴注PCV7，PCV7+OVA组及OVA组用OVA（卵清蛋白）致敏与激发。流式细胞术检测小鼠肺MHC-Ⅱ, CD40,CD80, CD86和纵隔淋巴结（MLN）MHC-Ⅱ, CD40, CD80, CD86的表达。ELISA检测BALF中INF-γ、IL-4、IL-5、IL-13、IL-17A、TGF-β及IL-10；BALB/c小鼠4周处死，分离得到骨髓单个核细胞，经GM-CSF和IL-4刺激得到骨髓来源的DCs，培养至第7天，阴性对照组加PBS，PCV7组加PCV7，阳性对照OVA组加OVA刺激DCs成熟，培养至第9天，收集未贴壁细胞用于形态学鉴定、流式细胞术分析，细胞上清用于ELISA检测。结果：PCV7+OVA组小鼠肺MHC-Ⅱ, CD40, CD86和纵隔淋巴结MHC-Ⅱ, CD40, CD86, CD80的表达较OVA组明显降低。PCV7+OVA组BALF中IL-13、IL-17A显著低于OVA组，而IFN-γ、IL-10显著高于OVA组；PCV7刺激DCs形态与OVA刺激DCs比较呈不成熟状态，PCV7刺激DCs MHC-Ⅱ, CD40, CD86表达较OVA刺激DCs明显降低，PCV7刺激DCs培养上清中细胞因子IL-12p70、IL-6较OVA刺激DCs明显降低，而TGF-β较OVA刺激DCs明显升高。结论：婴儿期PCV7免疫预防成年早期哮喘的可能机制是抑制DCs成熟增加Treg表达。
[Abstract]:Background and purpose
Bronchial asthma (asthma, asthma) is the most common chronic airway inflammatory disease. Its incidence is on the rise. At present, the way of clinical intervention can only control asthma symptoms. People have been trying to find effective asthma prevention and treatment methods.
Research shows that CpG-ODNs and BCG can inhibit allergic airway inflammation in a mouse model, but CpG-ODN treatment can cause harmful side effects, BCG is invalid on allergic asthma in human research. Streptococcus pneumoniae is a common pathogen of respiratory tract, can cause pneumonia, otitis media, meningitis and septicemia. Epidemiological studies have found that Streptococcus pneumoniae with a 7 valent vaccine (PCV7) can reduce the incidence of children and adults with asthma and related hospitalization rate. Studies have also shown that the vaccine can promote Treg and inhibit Th2 cells inhibit asthma features in adult mice. The PCV7 for infant immunization of children of Streptococcus pneumoniae infection, whether infants PCV7 can change the immune early adult experimental asthma CD4+T cell subsets and inhibit allergic airway inflammation is unclear.
Dendritic cells (DCs) are professional antigen-presenting cells, can activate CD4+T cells, can induce immune responses, and participate in immune tolerance, and the DCs function of regulating DCs function state contribute to the treatment and prevention of asthma. DCs in myeloid IL-4, granulocyte macrophage colony stimulating factor (GM-CSF) formation of immature under the induction of DCs, the antigen stimulated into mature DCs expressed high levels of CD80, CD86, CD40 and costimulatory molecules. Our previous study found that BCG vaccine can induce DCs mature, mature DCs promotes the expression of immune tolerance by increasing participation in Treg. DCs may be involved in regulating the immune PCV7 anti asthma.
The purpose of this study is to explore the effect and mechanism of PCV7 immunization on early adult experimental asthma in infancy, and to provide a new way of intervention for the prevention and treatment of asthma.
Part one infantile PCV7 immunization for early adult experimental asthma
Airway inflammation in mice and the effect of CD4+T cell subclass
Background: Streptococcus pneumoniae is the first bacterial pathogens in children with community acquired respiratory tract infections, WHO is recommended for children PCV7 vaccination against pneumococcal disease, PCV7 can significantly reduce the number of asthma attacks in children, reduce the severity of asthma, PCV7 can prevent asthma. The vaccine can promote the production of Treg and inhibition of Th2 cell inhibition characteristic of asthma performance in adult mice. The PCV7 for infant immunization of children of Streptococcus pneumoniae infection, whether infants can change PCV7 immune early adult CD4+T cell subsets and inhibit allergic airway inflammation is unclear. This study of infant immunization against PCV7 adult experimental early airway inflammation in asthmatic mice and CD4+T cells subgroup effects, lay the foundation for later study.
Objective: To investigate the effect of PCV7 immunization at infancy on airway inflammation and CD4+T cell subclass in early adult experimental asthma mice.
Methods: male 3 week BALB/c mice were randomly divided into control group (Control group), PCV7 immune experimental asthma group (PCV7+OVA group), asthma group (OVA group), PCV7+OVA group 3 weeks nasal instillation of PCV7, PCV7+OVA group and OVA group with OVA (ovalbumin) sensitized and excited. Rat pulmonary function tester in airway hyperresponsiveness; to observe the pathological changes of airway and lung tissue of mice HE staining; optical mice bronchoalveolar lavage fluid leukocyte count ELISA microscopy; detection of BALF in IL-4, IL-5, INF- gamma, IL-13, IL-17A, TGF- and beta IL-10; flow cytometry was used to detect CD4+Th cell subsets group expression.
Results: PCV7+OVA group of asthma symptoms in mice compared with the OVA group; airway hyperresponsiveness was significantly lower than that in OVA group; airway inflammation significantly reduced compared with OVA group.PCV7+OVA group BALF in total cells, eosinophils, neutrophils was significantly reduced compared with OVA group, IL-13 PCV7+OVA group BALF, IL-17A was significantly lower than group OVA, and IFN- gamma, IL-10 was significantly higher than that of OVA group, CD4+CD25+Foxp3+Treg group of mediastinal lymph node PCV7+OVA, CD4+INF- y +T cells was significantly higher than that of group OVA and CD4+IL-4+T of CD4+IL-17+T cells was significantly lower than that of OVA group.
Conclusion: PCV7 immunization in infancy is an effective way to prevent experimental asthma in early adult BALB/c mice.
The mechanism of the effect of PCV7 immunization on DCS in the second part
Background: PCV7 can promote infant immune CD4+CD25+Foxp3+Treg, Th1 cells, Th17 cells, inhibition of Th2, for the prevention of asthma in early adulthood, but the mechanism is not clear. Dendritic cells (DCs) are professional antigen-presenting cells, can activate CD4+T cells, can induce immune responses, and participate in the immune tolerance, and DCs the function of regulating the function of DCs state contribute to the prevention and treatment of asthma. DCs in myeloid IL-4, granulocyte macrophage colony stimulating factor (GM-CSF) formation of immature DCs induced by the antigen stimulated into mature DCs expressed high levels of MHC II and CD80, CD86, CD40 and costimulatory molecules.DCs uptake if PCV7 can reduce the expression of costimulatory molecules in the immature or mature state regulation can participate in immune tolerance, so the prevention and treatment of asthma. This part from two aspects of in vivo and in vitro study of PCV7 on DCs The effect of maturity is to explore the possible mechanism of PCV7 immunization in infancy to prevent early adult asthma, and to provide a new way of intervention for the prevention and treatment of asthma.
Objective: To study the effect of PCV7 on DCs in vivo and in vitro, and to explore the possible mechanism of PCV7 to prevent asthma.
Methods: male 3 week BALB/c mice were divided into control group (Control group), PCV7 immune experimental asthma group (PCV7+OVA group), asthma group (OVA group), PCV7+OVA group 3 weeks nasal instillation of PCV7, PCV7+OVA group and OVA group with OVA (ovalbumin) sensitization and excitation. The detection of lung MHC- mice II, flow cytometry CD40, CD80, CD86 and mediastinal lymph node (MLN) of MHC-, CD40, CD80, BALF CD86.ELISA was used to detect the expression of INF- in IL-4, IL-5, gamma, IL-13, IL-17A, TGF- and beta IL-10; BALB/c mice were sacrificed at 4 weeks, isolated bone marrow mononuclear cells. After GM-CSF and IL-4 stimulation by DCs from bone marrow, cultured for seventh days, the negative control group plus PBS, PCV7 plus PCV7 group, positive control group OVA plus OVA stimulated DCs maturation and cultured for ninth days, collect the non adherent cells were used for morphological identification, flow cytometry, cell supernatant for ELISA detection.
Results: PCV7+OVA group of mice lung MHC- II, CD40, CD86 and mediastinal lymph node MHC- II, CD40, CD86, CD80 expression was significantly lower than that of OVA group IL-13.PCV7+OVA group BALF, IL-17A was significantly lower than that of OVA group, and IFN- gamma, IL-10 was significantly higher than that of OVA group; PCV7 DCs and OVA DCs form of stimulus stimulus were not mature state, PCV7 stimulated DCs MHC- II, CD40, CD86 expression in OVA stimulated DCs decreased significantly, PCV7 stimulation of DCs cytokines in culture supernatants of IL-12p70, IL-6 with OVA stimulation DCs decreased significantly, while TGF- DCs was significantly higher than OVA beta stimulation.
Conclusion: the possible mechanism of PCV7 immunization in infancy to prevent early adult asthma is to inhibit the maturation of DCs and increase the expression of Treg.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R562.25

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