本文選題：哮喘　切入點(diǎn)：中性粒細(xì)胞　出處：《廣西醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：支氣管哮喘(哮喘)是多種細(xì)胞參與的慢性氣道炎癥,長期以來嗜酸性粒細(xì)胞被認(rèn)為是哮喘最重要的炎癥效應(yīng)細(xì)胞,但隨著哮喘氣道炎癥的深入研究,發(fā)現(xiàn)存在嗜酸細(xì)胞性哮喘(EA)及非嗜酸細(xì)胞性哮喘兩種不同的亞型,非嗜酸細(xì)胞性哮喘中半數(shù)以上為中性粒細(xì)胞性哮喘(NA),我們前期對哮喘患者的研究發(fā)現(xiàn)哮喘氣道中性粒細(xì)胞炎癥與氣道中性粒細(xì)胞凋亡障礙有關(guān),其發(fā)生機(jī)制仍在探討中。 哮喘發(fā)病機(jī)制的研究很大程度上需借助動物模型來進(jìn)行,但既往哮喘的動物研究多采用傳統(tǒng)的嗜酸細(xì)胞性哮喘動物模型,并不能真正反映中性粒細(xì)胞性哮喘的氣道炎癥狀態(tài)及氣道高反應(yīng)的特征。因此,本研究采用脂多糖聯(lián)合卵清蛋白氣道滴入致敏,卵清蛋白霧化激發(fā)建立中性粒細(xì)胞性哮喘小鼠模型。結(jié)果顯示小鼠出現(xiàn)了類似哮喘發(fā)作的癥狀及病理改變,以中性粒細(xì)胞為主的氣道炎癥,與嗜酸細(xì)胞性哮喘模型相似的氣道粘液高分泌及更嚴(yán)重的氣道高反應(yīng)(AHR)；提示該方法成功地建立了中性粒細(xì)胞性哮喘小鼠模型,為后續(xù)研究奠定了基礎(chǔ)。 目前認(rèn)為TH1/TH2細(xì)胞失衡所致TH2細(xì)胞優(yōu)勢是介導(dǎo)嗜酸細(xì)胞性哮喘發(fā)病最重要的免疫學(xué)機(jī)制,但最近的研究表明TH17細(xì)胞作為一類新發(fā)現(xiàn)的輔助性T細(xì)胞也參與哮喘發(fā)病。TH17細(xì)胞能夠分泌多種細(xì)胞因子,其中最重要的是IL-17A(亦稱IL-17), IL-17通過受體介導(dǎo)的信號途徑可誘導(dǎo)多種細(xì)胞產(chǎn)生IL-8、IL-6、GM-CSF、CXCL等細(xì)胞因子,趨化并激活中性粒細(xì)胞在局部浸潤�；赥H17細(xì)胞及IL-17受體信號途徑所產(chǎn)生的細(xì)胞因子對中性粒細(xì)胞的生物學(xué)功能,我們推測TH17細(xì)胞參與了中性粒細(xì)胞性哮喘發(fā)病,然而也有研究顯示TH17細(xì)胞參與了嗜酸細(xì)胞性哮喘發(fā)病。這兩種免疫學(xué)機(jī)制是分別或共同參與中性粒細(xì)胞性哮喘、嗜酸細(xì)胞性哮喘發(fā)病? 本研究結(jié)果顯示兩種哮喘小鼠脾臟TH17細(xì)胞比例、TH2細(xì)胞比例均高于對照組,提示TH17、TH2細(xì)胞均參與NA、EA小鼠發(fā)�。贿M(jìn)一步分析TH2/TH17比例,我們發(fā)現(xiàn)NA組低于EA組,且NA組支氣管肺泡灌洗液(BALF)中IL-17水平高于EA組,而IL-5水平明顯低于EA組,提示NA組小鼠產(chǎn)生IL-17的功能亢進(jìn),存在強(qiáng)烈的TH17細(xì)胞免疫反應(yīng)、中度TH2細(xì)胞免疫反應(yīng),TH17細(xì)胞在中性粒細(xì)胞性哮喘小鼠發(fā)病中起著更重要的作用。同時研究結(jié)果還表明增高的IL-6、TGF-β形成了有利于中性粒細(xì)胞性哮喘小鼠TH17細(xì)胞優(yōu)勢的體內(nèi)微環(huán)境,正調(diào)控TH17細(xì)胞分化的特異性轉(zhuǎn)錄因子RORyt的表達(dá)增強(qiáng)、負(fù)調(diào)控TH17細(xì)胞分化的SOCS3表達(dá)下調(diào)與中性粒細(xì)胞性哮喘小鼠體內(nèi)初始T細(xì)胞向TH17方向分化增強(qiáng)有關(guān)；而SOCS3表達(dá)增強(qiáng)是形成嗜酸細(xì)胞性哮喘小鼠TH2細(xì)胞優(yōu)勢的機(jī)制之一。 現(xiàn)有的免疫學(xué)理論認(rèn)為,激活的T細(xì)胞再次遇到刺激原時可誘導(dǎo)其進(jìn)入凋亡途徑,即激活誘導(dǎo)細(xì)胞死亡(activation-induced cell death, AICD),藉此限制免疫應(yīng)答的過度發(fā)展,AICD過程中任何異常都有可能造成機(jī)體的不正常表現(xiàn)甚至疾病。因此,已分化TH17細(xì)胞的凋亡與存活狀態(tài)很可能會影響哮喘氣道中性粒細(xì)胞炎癥的發(fā)生、發(fā)展。我們的研究發(fā)現(xiàn)NA組和EA組小鼠脾臟TH17細(xì)胞Ki-67的表達(dá)均明顯高于對照組,說明哮喘小鼠體內(nèi)已分化的TH17細(xì)胞的凋亡受到抑制,處于持續(xù)存活狀態(tài),因此,哮喘小鼠體內(nèi)除初始T細(xì)胞向TH17方向分化增強(qiáng)外,TH17細(xì)胞持續(xù)存活是導(dǎo)致其TH17細(xì)胞優(yōu)勢的另一重要機(jī)制。 白介素-7(IL-7)主要的功能是促進(jìn)B和T細(xì)胞生長、T細(xì)胞存活及增殖。哮喘小鼠TH17細(xì)胞持續(xù)存活的機(jī)制是否依賴于IL7-IL7受體信號途徑?本研究檢測了脾臟及BALF中IL-7的表達(dá),發(fā)現(xiàn)兩種哮喘小鼠模型IL-7表達(dá)均增高,高表達(dá)的IL-7提供了延長TH17細(xì)胞存活的微環(huán)境。與對照組相比,哮喘小鼠脾臟TH17細(xì)胞上STAT5的表達(dá)均明顯增高,且NA組高于EA組,提示哮喘小鼠TH17細(xì)胞存活依賴于JAK/STAT5信號途徑激活,且中性粒細(xì)胞性哮喘小鼠TH17細(xì)胞STAT5信號途徑激活程度更高；NA組TH17細(xì)胞上BCL-2的表達(dá)增高,提示中性粒細(xì)胞性哮喘小鼠TH17細(xì)胞通過上調(diào)抗凋亡蛋白BCL-2,抑制TH17細(xì)胞凋亡；兩組哮喘小鼠脾臟TH17細(xì)胞上凋亡終末剪切酶Caspase-3的表達(dá)均明顯增高,說明TH17的細(xì)胞凋亡是依賴Caspase途徑的細(xì)胞凋亡,在啟動抗凋亡機(jī)制的同時也激活了凋亡途徑；但Caspase-3上升的幅度明顯低于BCL-2,表明在促凋亡與抗凋亡的相互作用下抗凋亡的作用更強(qiáng)以致TH17細(xì)胞持續(xù)存活。IL-7信號通路可以被一些負(fù)調(diào)控機(jī)制所干擾,如SOCS-1作為細(xì)胞因子信號抑制劑可抑制STAT5信號轉(zhuǎn)導(dǎo)而維持適度的炎性反應(yīng),本研究發(fā)現(xiàn)兩種哮喘小鼠SOCS1表達(dá)均下調(diào),可能使機(jī)體出現(xiàn)IL-7信號的高反應(yīng)性,導(dǎo)致TH17細(xì)胞長期存活。 這些功能增強(qiáng)的TH17細(xì)胞如何參與哮喘氣道中性粒細(xì)胞炎癥的形成?與以往研究一致,本研究中兩種哮喘小鼠BALF中IL-17水平均增高；此外趨化因子CXCL1、CXCL5、CXCL8/IL-8也明顯增高且與IL-17水平呈正相關(guān),提示TH17細(xì)胞可能通過IL-17誘導(dǎo)趨化因子的表達(dá),募集中性粒細(xì)胞至氣道炎癥局部聚集。與我們在哮喘患者的研究一致,中性粒細(xì)胞性哮喘小鼠氣道中性粒細(xì)胞增高與氣道中性粒細(xì)胞凋亡受到抑制有關(guān)；此外我們還證實哮喘小鼠BALF中IL-8水平與AHR呈正相關(guān)。因此,TH17細(xì)胞可能通過IL-17促進(jìn)多種細(xì)胞因子分泌參與哮喘氣道中性粒細(xì)胞炎癥及AHR形成。 糖皮質(zhì)激素是目前控制哮喘氣道炎癥最有效的一線藥物,通過全身應(yīng)用地塞米松干預(yù)后肺組織病理改變減輕,哮喘小鼠BALF細(xì)胞總數(shù)、中性粒細(xì)胞均明顯下降,但仍然高于對照組；TH17細(xì)胞數(shù)量及相關(guān)細(xì)胞因子IL-17水平降低,說明地塞米松通過下調(diào)TH17細(xì)胞數(shù)量及相關(guān)細(xì)胞因子水平以減輕哮喘氣道中性粒細(xì)胞炎癥。但地塞米松不影響NA小鼠氣道中性粒細(xì)胞凋亡率,也未能影響兩種哮喘小鼠TH17細(xì)胞存活延長的狀態(tài)。 綜上所述,本研究成功建立了中性粒細(xì)胞性哮喘小鼠模型,在此基礎(chǔ)上證實中性粒細(xì)胞性哮喘小鼠存在強(qiáng)烈的TH17細(xì)胞免疫、中度TH2細(xì)胞免疫反應(yīng),增高的IL-6、TGF-β形成了有利于中性粒細(xì)胞性哮喘小鼠TH17細(xì)胞優(yōu)勢的體內(nèi)微環(huán)境,并通過RORγt和SOCS3兩種途徑共同調(diào)控TH17細(xì)胞的優(yōu)勢分化。除初始T細(xì)胞向TH17方向分化增強(qiáng)外,中性粒細(xì)胞性哮喘小鼠體內(nèi)已分化的TH17細(xì)胞在機(jī)體高IL-7微環(huán)境下,依賴JAK/STAT5信號途徑激活維持其存活狀態(tài),高表達(dá)的IL-17通過促進(jìn)多種細(xì)胞因子分泌參與哮喘氣道中性粒細(xì)胞炎癥及AHR形成。地塞米松下調(diào)TH17細(xì)胞數(shù)量及相關(guān)細(xì)胞因子水平是減輕哮喘氣道中性粒細(xì)胞炎癥的機(jī)制之一；但地塞米松對氣道中性粒細(xì)胞凋亡率及TH17細(xì)胞存活延長的狀態(tài)無明顯影響,可能是哮喘激素抵抗的免疫學(xué)機(jī)制之一。本研究初步闡明TH17細(xì)胞及其功能狀態(tài)在哮喘發(fā)病中的作用及相關(guān)分子機(jī)制,為尋找哮喘治療靶點(diǎn)提供了新線索和理論依據(jù)。
[Abstract]:Bronchial asthma (asthma) is a chronic airway inflammation in various cells, long-term since eosinophils are thought to be the most important effector cells in asthma, but with the in-depth study of airway inflammation in asthma, found eosinophilic asthma (EA) and non eosinophilic asthma in two different subtypes and non eosinophilic asthma in more than half of neutrophilic asthma (NA), we study on the early asthmatic patients found that airway neutrophilic inflammation is associated with airway neutrophil apoptosis, its mechanism is still under discussion.
Study on the pathogenesis of asthma is largely to use animal models, but animal studies asthma use more eosinophilic asthma animal model of tradition, and can not truly reflect the neutrophilic asthma airway inflammation and airway hyperresponsiveness characteristics. Therefore, this study uses lipopolysaccharide and airway instillation of ovalbumin sensitized ovalbumin inhalation to establish a mouse model of neutrophilic asthma. Results showed that the mice developed asthma like symptoms and pathological changes of airway inflammation in neutrophils, similar to eosinophilic asthma airway mucus hypersecretion and more severe airway hyperresponsiveness (AHR) showed the method; successfully established a mouse model of neutrophilic asthma, laid the foundation for further study.
Now that the TH1/TH2 cells caused by the imbalance of TH2 cells is mediated by the advantage of eosinophilic asthma most important immunological mechanisms, but recent studies suggest that TH17 cells as a kind of new T helper cells are found in.TH17 cells of asthma can secrete a variety of cytokines, the most important of which is IL-17A (also called IL-17). IL-17 signaling pathway mediated by receptors can produce IL-8, many cells induced IL-6, GM-CSF, CXCL and other cytokines, chemotaxis and activation of neutrophil infiltration in the local. The biological function of TH17 cells and IL-17 receptor signaling pathway based on cytokines produced by the neutrophil, we speculate that TH17 cells involved in the pathogenesis of neutrophilic asthma, however, studies have shown that TH17 cells are involved in the pathogenesis of eosinophilic asthma. Addicted to these two kinds of immunological mechanisms are respectively or jointly participate in neutrophilic asthma Asthma, eosinophilic asthma?
The results of this study showed that two kinds of asthma mice spleen TH17 cell ratio, TH2 cell ratio was higher than the control group, suggesting that TH17 cells are involved in NA, TH2, EA of mouse disease; further analysis of the proportion of TH2/TH17, we found that NA group than in EA group, NA group and bronchoalveolar lavage fluid (BALF) IL-17 levels higher than the EA group. While the IL-5 level was significantly lower than that of group EA, suggesting that NA mice hyperfunction of IL-17, TH17 cells in presence of a strong immune response, immune TH2 cells TH17 cells in response to moderate, neutrophils in the pathogenesis of asthma mice plays a more important role. The results also showed that the increased IL-6, TGF- beta form in favor of neutrophilic asthma mice TH17 cells in vivo microenvironment, enhance the positive regulation of RORyt expression of specific transcription factor of TH17 cell differentiation, negative regulation of TH17 cell differentiation by down-regulation of SOCS3 expression and neutrophil In asthmatic mice, the initial T cells were enhanced to differentiate into TH17, and SOCS3 expression was one of the mechanisms of TH2 cell dominance in eosinophilic asthmatic mice.
Think that the existing theory of immunology, the activation of T cells again encountered stimuli can induce the apoptosis in ways that activation induced cell death (activation-induced cell, death, AICD), so as to limit the excessive development of the immune response, AICD in the process of any anomalies may be caused by the body is not normal and disease. Therefore, has the differentiation of TH17 cell apoptosis and survival status may affect airway neutrophil inflammation. We found that the expression of NA group and EA group of mice spleen TH17 cells of Ki-67 were significantly higher than the control group, indicating apoptosis in asthmatic mice differentiated TH17 cells was inhibited, in a sustained survival state. Therefore, in addition to the initial T cells in asthmatic mice to TH17 differentiation increased, TH17 cell survival is another important mechanism leading to the TH17 cells.
Interleukin -7 (IL-7) the main function is to promote the growth of B and T cells, T cell survival and proliferation. The mechanism of TH17 cells in asthmatic mice continue to survive depends on IL7-IL7 receptor signal pathway? This study examined the expression of IL-7 in spleen and BALF, found that two kinds of mouse model of asthma IL-7 expression increased, high expression the IL-7 provides extended microenvironment for TH17 cell survival. Compared with the control group, TH17 cells of asthmatic mice spleen STAT5 expression were significantly increased, and the NA group than in EA group, suggesting that TH17 cells of asthmatic mice survival depends on JAK/STAT5 signaling pathway activation, and neutrophilic asthma mice TH17 cell activation of STAT5 signaling pathway a higher degree; the expression of BCL-2 NA in TH17 cells, suggesting that neutrophils of asthmatic mice TH17 cells by up regulation of anti apoptotic protein BCL-2, inhibiting the apoptosis of TH17 cells; two groups of spleen TH17 in asthmatic mice The expression of apoptosis cells end shear enzyme Caspase-3 were significantly increased, indicating TH17 apoptosis is Caspase dependent apoptosis pathway, at the start of the anti apoptosis mechanism also activate apoptosis pathway; but the Caspase-3 increase was significantly lower than that of BCL-2, indicated that in the pro apoptotic and anti apoptotic interactions of anti apoptosis a stronger effect that TH17 cells survive.IL-7 signaling pathway may be some interference of negative regulatory mechanism, such as SOCS-1 cytokine signaling inhibitors can inhibit STAT5 signaling and maintain moderate inflammatory reaction, the study found that two of asthmatic mice SOCS1 expression were down regulated, high reactivity may make the body appear IL-7 signals, resulting in long-term the survival of TH17 cells.
榪欎簺鍔熻兘澧炲己鐨凾H17緇嗚優(yōu)濡備綍鍙備笌鍝枠姘旈亾涓，

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