本文關(guān)鍵詞： 嬰兒期PCV7免疫 實驗性哮喘 成年早期 Th2 Th17 Foxp3+Treg 不成熟DCs 成年早期哮喘 嬰兒期PCV7免疫　出處：《重慶醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：背景和目的 支氣管哮喘(哮喘，asthma)是最常見的慢性氣道炎癥性疾病，其發(fā)病率呈持續(xù)上升趨勢。目前臨床干預(yù)哮喘的方法都只能控制哮喘癥狀，人們一直在努力尋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過敏性氣道炎癥，，然而CpG-ODN治療可以引起有害的副作用，BCG在人類的研究中對過敏性哮喘無效。肺炎鏈球菌是一種常見的呼吸道病原，可以引起肺炎、中耳炎、腦膜炎和敗血癥。流行病學(xué)研究發(fā)現(xiàn)肺炎鏈球菌7價結(jié)合疫苗（PCV7）可以減少兒童及成人哮喘的發(fā)病率及相關(guān)住院率。也有研究表明該疫苗在成年期小鼠免疫可以通過促進(jìn)Treg和抑制Th2細(xì)胞的產(chǎn)生抑制哮喘的特征性表現(xiàn)。目前PCV7用于嬰兒期免疫預(yù)防兒童肺炎鏈球菌感染，是否嬰兒期PCV7免疫可以改變成年早期實驗性哮喘CD4+T細(xì)胞亞類及抑制變應(yīng)性氣道炎癥還不清楚。 樹突狀細(xì)胞（DCs）是專職抗原呈遞細(xì)胞，能激活初始CD4+T細(xì)胞，既誘導(dǎo)免疫反應(yīng)，又參與免疫耐受，這與DCs功能狀態(tài)有關(guān)，調(diào)節(jié)DCs功能狀態(tài)有助于治療和預(yù)防哮喘。髓樣DCs在IL-4、粒巨噬細(xì)胞集落刺激因子(GM-CSF)誘導(dǎo)下形成不成熟DCs，經(jīng)抗原刺激后變?yōu)槌墒霥Cs，表達(dá)高水平CD80、CD86、CD40等共刺激分子。我們前期研究發(fā)現(xiàn)疫苗BCG能誘導(dǎo)DCs部分成熟，部分成熟DCs促進(jìn)Treg表達(dá)增加參與免疫耐受。提示DCs可能參與了PCV7抗哮喘的免疫調(diào)節(jié)。 本研究旨在探討嬰兒期PCV7免疫對成年早期實驗性哮喘的影響及作用機(jī)制，為哮喘防治提供新的干預(yù)思路。 第一部分嬰兒期PCV7免疫對成年早期實驗性哮喘 小鼠氣道炎癥、CD4+T細(xì)胞亞類的影響 背景：肺炎鏈球菌是兒童社區(qū)獲得性呼吸道感染的第一位細(xì)菌病原體，WHO推薦兒童接種PCV7預(yù)防肺炎鏈球菌疾病， PCV7能明顯減少兒童哮喘發(fā)作次數(shù)、減輕哮喘嚴(yán)重程度，PCV7能預(yù)防哮喘。該疫苗在成年期小鼠免疫可以通過促進(jìn)Treg的產(chǎn)生和抑制Th2細(xì)胞的產(chǎn)生抑制哮喘的特征性表現(xiàn)。目前PCV7用于嬰兒期免疫預(yù)防兒童肺炎鏈球菌感染，是否嬰兒期PCV7免疫可以改變成年早期CD4+T細(xì)胞亞類及抑制過敏性氣道炎癥還不清楚。因此本研究探討嬰兒期PCV7免疫對成年早期實驗性哮喘小鼠氣道炎癥及CD4+T細(xì)胞亞類的影響，為后期研究奠定基礎(chǔ)。 目的：探討嬰兒期PCV7免疫對成年早期實驗性哮喘小鼠氣道炎癥及CD4+T細(xì)胞亞類的影響。 方法：清潔級3周BALB/c小鼠隨機(jī)分為對照組（Control組）、PCV7免疫實驗性哮喘組（PCV7+OVA組）、實驗性哮喘組（OVA組），PCV7+OVA組3周時鼻腔滴注PCV7，PCV7+OVA組及OVA組用OVA（卵清蛋白）致敏與激發(fā)。小鼠肺功能儀測定小鼠氣道高反應(yīng)性；HE染色觀察小鼠氣道及肺組織病理變化；光學(xué)顯微鏡觀察小鼠支氣管肺泡灌洗液白細(xì)胞分類計數(shù)；ELISA檢測BALF中INF-γ、IL-4、IL-5、IL-13、IL-17A、TGF-β及IL-10；流式細(xì)胞術(shù)檢測CD4+Th細(xì)胞各亞群表達(dá)情況。 結(jié)果：PCV7+OVA組小鼠哮喘癥狀較OVA組減輕；氣道高反應(yīng)性顯著低于OVA組小鼠；氣道炎癥較OVA組明顯減輕。PCV7+OVA組BALF中細(xì)胞總數(shù)、嗜酸性粒細(xì)胞、中性粒細(xì)胞較OVA組明顯減少，PCV7+OVA組BALF中IL-13、IL-17A顯著低于OVA組，而IFN-γ、IL-10顯著高于OVA組， PCV7+OVA組縱隔淋巴結(jié)CD4+CD25+Foxp3+Treg、 CD4+INF-γ+T細(xì)胞顯著高于OVA組，而CD4+IL-4+T、CD4+IL-17+T細(xì)胞顯著低于OVA組。 結(jié)論：嬰兒期PCV7免疫是預(yù)防成年早期BALB/c小鼠實驗性哮喘的有效方法。 第二部分PCV7免疫對DCS影響的機(jī)制研究 背景：嬰兒期PCV7免疫可以促進(jìn)CD4+CD25+Foxp3+Treg、Th1細(xì)胞產(chǎn)生，抑制Th2、Th17細(xì)胞產(chǎn)生，用于預(yù)防成年早期哮喘，但其機(jī)制尚不清楚。樹突狀細(xì)胞（DCs）是專職抗原呈遞細(xì)胞，能激活初始CD4+T細(xì)胞，既誘導(dǎo)免疫反應(yīng)，又參與免疫耐受，這與DCs功能狀態(tài)有關(guān)，調(diào)節(jié)DCs功能狀態(tài)有助于治療和預(yù)防哮喘。髓樣DCs在IL-4、粒巨噬細(xì)胞集落刺激因子(GM-CSF)誘導(dǎo)下形成不成熟DCs，經(jīng)抗原刺激后變?yōu)槌墒霥Cs，表達(dá)高水平MHCⅡ和CD80、CD86、CD40等共刺激分子。DCs攝取PCV7后如果共刺激分子的表達(dá)能夠降低處于未成熟或部分成熟狀態(tài)即可參與免疫耐受的調(diào)節(jié)，從而預(yù)防和治療哮喘。本部分從體內(nèi)和體外兩個方面研究PCV7對DCs成熟度的影響，旨在探討嬰兒期PCV7免疫預(yù)防成年早期哮喘的可能發(fā)生機(jī)制，為哮喘防治提供新的干預(yù)思路。 目的：研究體內(nèi)和體外PCV7對DCs的影響，探討PCV7預(yù)防哮喘的可能發(fā)生機(jī)制。 方法：清潔級3周BALB/c小鼠分為對照組（Control組）、PCV7免疫實驗性哮喘組（PCV7+OVA組）、實驗性哮喘組（OVA組），PCV7+OVA組3周時鼻腔滴注PCV7，PCV7+OVA組及OVA組用OVA（卵清蛋白）致敏與激發(fā)。流式細(xì)胞術(shù)檢測小鼠肺MHC-Ⅱ, CD40,CD80, CD86和縱隔淋巴結(jié)（MLN）MHC-Ⅱ, CD40, CD80, CD86的表達(dá)。ELISA檢測BALF中INF-γ、IL-4、IL-5、IL-13、IL-17A、TGF-β及IL-10；BALB/c小鼠4周處死，分離得到骨髓單個核細(xì)胞，經(jīng)GM-CSF和IL-4刺激得到骨髓來源的DCs，培養(yǎng)至第7天，陰性對照組加PBS，PCV7組加PCV7，陽性對照OVA組加OVA刺激DCs成熟，培養(yǎng)至第9天，收集未貼壁細(xì)胞用于形態(tài)學(xué)鑒定、流式細(xì)胞術(shù)分析，細(xì)胞上清用于ELISA檢測。 結(jié)果：PCV7+OVA組小鼠肺MHC-Ⅱ, CD40, CD86和縱隔淋巴結(jié)MHC-Ⅱ, CD40, CD86, CD80的表達(dá)較OVA組明顯降低。PCV7+OVA組BALF中IL-13、IL-17A顯著低于OVA組，而IFN-γ、IL-10顯著高于OVA組；PCV7刺激DCs形態(tài)與OVA刺激DCs比較呈不成熟狀態(tài)，PCV7刺激DCs MHC-Ⅱ, CD40, CD86表達(dá)較OVA刺激DCs明顯降低，PCV7刺激DCs培養(yǎng)上清中細(xì)胞因子IL-12p70、IL-6較OVA刺激DCs明顯降低，而TGF-β較OVA刺激DCs明顯升高。 結(jié)論：嬰兒期PCV7免疫預(yù)防成年早期哮喘的可能機(jī)制是抑制DCs成熟增加Treg表達(dá)。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R562.25

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相關(guān)期刊論文 前1條

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