联合拮抗IL-13及IL-25对哮喘小鼠气道炎症及气道重塑抑制作用的研究

发布时间：2018-06-07 10:37

  本文选题：哮喘 + IL-13　； 参考：《第四军医大学》2017年博士论文

【摘要】：哮喘是一种气道的慢性炎症性疾病,由气道自身结构细胞和炎症细胞及其细胞组分共同参与。哮喘患者气道反复炎症以及持续重塑促进了不可逆性的肺功能下降,增加了对当前哮喘治疗药物的抵抗。传统观点认为Th2占优势适应性免疫应答在哮喘发生发展中起关键作用。Th2型细胞因子IL-4、IL-5及IL-13等与其靶细胞相互作用导致哮喘典型病理生理改变;其中IL-13在哮喘的气道炎症、气道高反应以及重塑中的作用已经得到肯定,机制也日趋明确。但目前临床上有关靶向阻断IL-13的哮喘治疗策略并没有明确获益。究其原因,一方面,Th2类细胞因子的功能效应间存在相互代偿;另一方面,适应性免疫应答与固有免疫应答之间存在复杂的交互。气道上皮层不仅作为结构屏障,其还可以对环境刺激做出应答,分泌相应的炎症介质IL-25、IL-33和TSLP等,激活粘膜周围ILC2细胞进而导致哮喘的发生发展。固有免疫应答作用的发现为哮喘基于肺部的发病机制与Th2型炎症提供了一个紧密的联系;并一定程度解释了靶向阻断以Th2为中心的适应性免疫应答并不能最大临床获益的原因。尽管IL-25、IL-33和TSLP均能诱导哮喘的发生,IL-33主要病毒相关疾病中高表达、TSLP主要在毒性疾病(如吸烟)高表达,而IL-25在变应性哮喘中表达较高,且IL-25可以促进IL-33以及TSLP在肺部的表达,说明IL-25在变应性哮喘中的作用更为突出。基于此,我们设想联合阻断适应性及固有免疫应答的关键因子IL-13和IL-25,可能会更好的下调哮喘的气道炎症和气道重塑。为此,进行了以下研究:方法:1.OVA诱导哮喘小鼠模型,BALF中细胞计数,HE染色、Masson染色观察模型制备情况。2.Real-Time PC R检测哮喘模型小鼠肺组织IL-13和IL-25 mRNA的表达情况,ELISA检测BALF中IL-13和IL-25的蛋白表达水平,免疫组化染色对肺组织IL-13和IL-25进行定位。3.应用sIL-13R和s IL-25R靶向拮抗哮喘小鼠IL-13和IL-25后,BALF中细胞计数,肺组织HE染色,观察气道炎症的变化。以Mch诱导各组小鼠,观察气道高反应的变化。4.联合拮抗IL-13和IL-25后,ELISA检测BALF以及肺组织匀浆中Th2型细胞因子IL-4、IL-5和IL-13表达水平,以及固有免疫细胞因子IL-25、IL-33和TSLP的表达水平。5.肺组织PAS染色,ELISA检测BALF及肺组织中MUC5AC糖蛋白表达水平,来反映联合拮抗IL-13和IL-25后气道杯状细胞的化生和黏液分泌状态。6.天狼星红染色、胶原检测来观察支气管周围纤维化情况;Western blotting检测TGF-β1,Smad2以及p-Smad2的蛋白水平,分析拮抗IL-13和IL-25抑制胶原产生的机制。7.抗α-SMA和PCNA免疫组化染色,观察联合拮抗IL-13和IL-25对气道平滑肌细胞肥大和增殖的影响。8.抗vWF免疫组化染色,ELISA检测BALF及肺组织VEGF表达水平,观察联合拮抗IL-13和IL-25对肺组织新生血管的影响。结果:1.OVA成功诱导哮喘小鼠模型产生,哮喘模型BALF中嗜酸细胞增高、肺组织炎症浸润明显、气道周围纤维化显著。哮喘模型IL-13和IL-25的mRNA及蛋白表达水平均显著上调。2.应用sIL-13R和s IL-25R靶向拮抗哮喘小鼠IL-13和IL-25后,BALF中嗜酸细胞显著减少,肺组织炎症细胞浸润明显减轻,哮喘小鼠气道高反应明显控制。3.联合拮抗IL-13和IL-25后,BALF及肺组织匀浆中Th2型细胞因子IL-4、IL-5和IL-13以及固有免疫细胞因子IL-25、IL-33和TSLP的表达水平均显著下降。4.联合拮抗IL-13和IL-25后,哮喘小鼠MUC5AC糖蛋白表达明显抑制,气道PAS评分及杯状细胞area/Pbm比例明显下降。5.联合拮抗IL-13和IL-25后,TGF-β1及p-Smad2的蛋白水平明显下调,进而导致气道周围胶原的堆积明显减轻。6.抗α-SMA和PCNA免疫组化染色结果显示,联合拮抗IL-13和IL-25后气道平滑肌细胞肥大和增殖明显受到抑制。7.联合拮抗IL-13和IL-25后,VEGF表达水平明显下调,抗vWF阳性血管明显减少。结论:1.用OVA致敏和激发成功构建哮喘小鼠模型,该模型中IL-13及IL-25的mRNA及蛋白水平均显著高于正常对照小鼠。2.联合拮抗IL-13和IL-25可显著抑制过敏原诱导的气道嗜酸细胞炎症、气道高反应以及适应性和固有免疫应答的相关炎症因子水平。3.联合拮抗IL-13和IL-25可显著抑制过敏原诱导的气道黏液高分泌、细胞外基质沉积、气道平滑肌增厚以及肺组织血管新生从而抑制哮喘气道重塑。有望为临床治疗哮喘提供一种新思路。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R562.25
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本文编号：1990877