本文選題：哮喘 + IL-13��； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：哮喘是一種氣道的慢性炎癥性疾病,由氣道自身結(jié)構(gòu)細(xì)胞和炎癥細(xì)胞及其細(xì)胞組分共同參與。哮喘患者氣道反復(fù)炎癥以及持續(xù)重塑促進了不可逆性的肺功能下降,增加了對當(dāng)前哮喘治療藥物的抵抗。傳統(tǒng)觀點認(rèn)為Th2占優(yōu)勢適應(yīng)性免疫應(yīng)答在哮喘發(fā)生發(fā)展中起關(guān)鍵作用。Th2型細(xì)胞因子IL-4、IL-5及IL-13等與其靶細(xì)胞相互作用導(dǎo)致哮喘典型病理生理改變;其中IL-13在哮喘的氣道炎癥、氣道高反應(yīng)以及重塑中的作用已經(jīng)得到肯定,機制也日趨明確。但目前臨床上有關(guān)靶向阻斷IL-13的哮喘治療策略并沒有明確獲益。究其原因,一方面,Th2類細(xì)胞因子的功能效應(yīng)間存在相互代償;另一方面,適應(yīng)性免疫應(yīng)答與固有免疫應(yīng)答之間存在復(fù)雜的交互。氣道上皮層不僅作為結(jié)構(gòu)屏障,其還可以對環(huán)境刺激做出應(yīng)答,分泌相應(yīng)的炎癥介質(zhì)IL-25、IL-33和TSLP等,激活粘膜周圍ILC2細(xì)胞進而導(dǎo)致哮喘的發(fā)生發(fā)展。固有免疫應(yīng)答作用的發(fā)現(xiàn)為哮喘基于肺部的發(fā)病機制與Th2型炎癥提供了一個緊密的聯(lián)系;并一定程度解釋了靶向阻斷以Th2為中心的適應(yīng)性免疫應(yīng)答并不能最大臨床獲益的原因。盡管IL-25、IL-33和TSLP均能誘導(dǎo)哮喘的發(fā)生,IL-33主要病毒相關(guān)疾病中高表達、TSLP主要在毒性疾病(如吸煙)高表達,而IL-25在變應(yīng)性哮喘中表達較高,且IL-25可以促進IL-33以及TSLP在肺部的表達,說明IL-25在變應(yīng)性哮喘中的作用更為突出�；诖�,我們設(shè)想聯(lián)合阻斷適應(yīng)性及固有免疫應(yīng)答的關(guān)鍵因子IL-13和IL-25,可能會更好的下調(diào)哮喘的氣道炎癥和氣道重塑。為此,進行了以下研究:方法:1.OVA誘導(dǎo)哮喘小鼠模型,BALF中細(xì)胞計數(shù),HE染色、Masson染色觀察模型制備情況。2.Real-Time PC R檢測哮喘模型小鼠肺組織IL-13和IL-25 mRNA的表達情況,ELISA檢測BALF中IL-13和IL-25的蛋白表達水平,免疫組化染色對肺組織IL-13和IL-25進行定位。3.應(yīng)用sIL-13R和s IL-25R靶向拮抗哮喘小鼠IL-13和IL-25后,BALF中細(xì)胞計數(shù),肺組織HE染色,觀察氣道炎癥的變化。以Mch誘導(dǎo)各組小鼠,觀察氣道高反應(yīng)的變化。4.聯(lián)合拮抗IL-13和IL-25后,ELISA檢測BALF以及肺組織勻漿中Th2型細(xì)胞因子IL-4、IL-5和IL-13表達水平,以及固有免疫細(xì)胞因子IL-25、IL-33和TSLP的表達水平。5.肺組織PAS染色,ELISA檢測BALF及肺組織中MUC5AC糖蛋白表達水平,來反映聯(lián)合拮抗IL-13和IL-25后氣道杯狀細(xì)胞的化生和黏液分泌狀態(tài)。6.天狼星紅染色、膠原檢測來觀察支氣管周圍纖維化情況;Western blotting檢測TGF-β1,Smad2以及p-Smad2的蛋白水平,分析拮抗IL-13和IL-25抑制膠原產(chǎn)生的機制。7.抗α-SMA和PCNA免疫組化染色,觀察聯(lián)合拮抗IL-13和IL-25對氣道平滑肌細(xì)胞肥大和增殖的影響。8.抗vWF免疫組化染色,ELISA檢測BALF及肺組織VEGF表達水平,觀察聯(lián)合拮抗IL-13和IL-25對肺組織新生血管的影響。結(jié)果:1.OVA成功誘導(dǎo)哮喘小鼠模型產(chǎn)生,哮喘模型BALF中嗜酸細(xì)胞增高、肺組織炎癥浸潤明顯、氣道周圍纖維化顯著。哮喘模型IL-13和IL-25的mRNA及蛋白表達水平均顯著上調(diào)。2.應(yīng)用sIL-13R和s IL-25R靶向拮抗哮喘小鼠IL-13和IL-25后,BALF中嗜酸細(xì)胞顯著減少,肺組織炎癥細(xì)胞浸潤明顯減輕,哮喘小鼠氣道高反應(yīng)明顯控制。3.聯(lián)合拮抗IL-13和IL-25后,BALF及肺組織勻漿中Th2型細(xì)胞因子IL-4、IL-5和IL-13以及固有免疫細(xì)胞因子IL-25、IL-33和TSLP的表達水平均顯著下降。4.聯(lián)合拮抗IL-13和IL-25后,哮喘小鼠MUC5AC糖蛋白表達明顯抑制,氣道PAS評分及杯狀細(xì)胞area/Pbm比例明顯下降。5.聯(lián)合拮抗IL-13和IL-25后,TGF-β1及p-Smad2的蛋白水平明顯下調(diào),進而導(dǎo)致氣道周圍膠原的堆積明顯減輕。6.抗α-SMA和PCNA免疫組化染色結(jié)果顯示,聯(lián)合拮抗IL-13和IL-25后氣道平滑肌細(xì)胞肥大和增殖明顯受到抑制。7.聯(lián)合拮抗IL-13和IL-25后,VEGF表達水平明顯下調(diào),抗vWF陽性血管明顯減少。結(jié)論:1.用OVA致敏和激發(fā)成功構(gòu)建哮喘小鼠模型,該模型中IL-13及IL-25的mRNA及蛋白水平均顯著高于正常對照小鼠。2.聯(lián)合拮抗IL-13和IL-25可顯著抑制過敏原誘導(dǎo)的氣道嗜酸細(xì)胞炎癥、氣道高反應(yīng)以及適應(yīng)性和固有免疫應(yīng)答的相關(guān)炎癥因子水平。3.聯(lián)合拮抗IL-13和IL-25可顯著抑制過敏原誘導(dǎo)的氣道黏液高分泌、細(xì)胞外基質(zhì)沉積、氣道平滑肌增厚以及肺組織血管新生從而抑制哮喘氣道重塑。有望為臨床治療哮喘提供一種新思路。
[Abstract]:Asthma is a chronic inflammatory disease of the airway, which is involved in the airway's own structural cells and inflammatory cells and their cell components. Repeated airway inflammation and continuous remodeling in asthmatic patients promote the decline of the irreversible lung function and increase the resistance to current asthma treatment. The traditional view is that Th2 is the dominant adaptive immune system. Response to the development of asthma, the key role of.Th2 cytokine IL-4, IL-5 and IL-13, and their interaction with the target cells leads to typical pathophysiological changes in asthma; the role of IL-13 in airway inflammation, airway hyperresponsiveness and remodeling has been affirmed, and the mechanism is becoming increasingly clear. On the one hand, there is a complex interaction between the adaptive immune response and the inherent immune response. On the other hand, there is a complex interaction between the adaptive immune response and the inherent immune response. The airway epithelium is not only a structural barrier, but also responds to environmental stimuli, and is secreted by the IL-13. The corresponding inflammatory mediators, such as IL-25, IL-33 and TSLP, activate the ILC2 cells around the mucosa and lead to the development of asthma. The discovery of the inherent immune response provides a close association with the Th2 type of inflammation based on the pathogenesis of asthma based on the lung, and to a certain extent explains the adaptive immune response targeting the target blocking Th2 centered. Although IL-25, IL-33 and TSLP can induce asthma, high expression of IL-33 major virus related diseases, TSLP is highly expressed in toxic diseases such as smoking, and IL-25 is highly expressed in allergic asthma, and IL-25 can promote the expression of IL-33 and TSLP in the lungs, indicating that IL-25 is in allergic asthma. Based on this, we envisage a combination of IL-13 and IL-25, a key factor in blocking adaptation and inherent immune response, which may better reduce airway inflammation and airway remodeling in asthma. The following studies are: 1.OVA induced asthma mice model, cell count in BALF, HE staining, and Masson staining observation model .2.Real-Time PC R was used to detect the expression of IL-13 and IL-25 mRNA in the lung tissue of the asthmatic model mice. The protein expression level of IL-13 and IL-25 in BALF was detected by ELISA. The immunohistochemical staining was used to locate the lung tissue IL-13 and IL-25. HE staining was used to observe the changes in airway inflammation. The mice were induced by Mch, and the changes in airway hyperactivity were observed by.4. combined with IL-13 and IL-25. ELISA was used to detect BALF and the IL-4, IL-5 and IL-13 expression of Th2 type cytokines in the lung homogenate, as well as the expression level of the intrinsic immune cell factor and the expression level of the lung tissue. The expression level of MUC5AC glycoprotein in BALF and lung tissue was detected by ELISA to reflect the metaplasia and mucus secretion of the goblet cells after the combination of IL-13 and IL-25,.6. Sirius red staining, and collagen detection was used to observe the fibrosis in the bronchi; Western blotting detected the protein levels of TGF- beta 1, Smad2 and p-Smad2, and analyzed the antagonistic IL-13. And IL-25 inhibition of collagen production mechanism.7. anti alpha -SMA and PCNA immunohistochemical staining, observe the effect of combined antagonism of IL-13 and IL-25 on the hypertrophy and proliferation of airway smooth muscle cells,.8. anti vWF immunohistochemical staining, ELISA detection BALF and lung tissue VEGF expression level, observe the effect of joint antagonistic IL-13 and negative on lung tissue neovascularization. Results: VA successfully induced the model of asthmatic mice, the eosinophil in the asthmatic model BALF, the inflammatory infiltration of the lung tissue, and the fibrosis in the airway, the mRNA and the protein expression level of the asthma model IL-13 and IL-25 all significantly up the.2. application sIL-13R and s IL-25R target to the IL-13 and IL-25 of the antagonistic asthmatic rats, and the eosinophils in the BALF were significantly reduced. The infiltration of inflammatory cells in lung tissue was significantly reduced. The airway hyperresponsiveness of asthmatic mice was obviously controlled by.3. combined with IL-13 and IL-25, and the IL-4, IL-5 and IL-13 as well as the expression level of Th2 and inherent immune cell factors in BALF and lung homogenate were significantly lower than those of.4., IL-33 and TSLP. The expression of C5AC glycoprotein was obviously inhibited. The PAS score of the airway and the proportion of area/Pbm in goblet cells decreased significantly by.5. combined with IL-13 and IL-25, and the protein levels of TGF- beta 1 and p-Smad2 decreased significantly, which led to the accumulation of collagen around the airway obviously alleviated the results of.6. anti alpha -SMA and PCNA immunohistochemical staining, and combined antagonism of IL-13 and post gas. The hypertrophy and proliferation of the smooth muscle cells were obviously inhibited by.7. combined with IL-13 and IL-25, the expression level of VEGF was obviously down, and the anti vWF positive blood vessels were obviously reduced. Conclusion: 1. the asthma mice model was successfully constructed by OVA sensitization and excitation. The mRNA and protein levels of IL-13 and IL-25 in this model were significantly higher than those of normal control mice. IL-13 and IL-25 significantly inhibit anaphylaxis induced eosinophil inflammation, airway hyperresponsiveness and adaptive and inherent immune response related inflammatory factors level.3. combined with antagonistic IL-13 and IL-25 can significantly inhibit allergen induced airway mucus hypersecretion, extracellular matrix deposition, airway smooth muscle thickening, and pulmonary vascular neovascularization. Therefore, it can inhibit airway remodeling in asthma, and is expected to provide a new way for clinical treatment of asthma.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R562.25
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本文編號：1990877