IFN-γ对小鼠急性过敏性气道炎症的抑制作用

发布时间：2018-06-17 11:31

本文选题：干扰素-γ + 2型固有淋巴细胞　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景过敏性气道炎症是过敏性哮喘的病理生理基础,由对外部刺激不适当的免疫应答引起。长期以来一直认为哮喘是由抗原诱导Th2细胞活化引起的。近期的研究表明2型固有淋巴细胞(Group 2 innate lymphoid cells,ILC2s)在哮喘的发生过程中发挥重要的作用。ILC2s广泛分布于脂肪相关淋巴集簇和包括皮肤、肝脏、肺、小肠、内脏脂肪组织在内的非淋巴组织。上皮来源的细胞因子胸腺基质淋巴细胞生成素(Thymic stromal lymphopoietin,TSLP)、白细胞介素-33(Interleukin-33,IL-33)、IL-25均可以活化ILC2s。IL-33是IL-1家族中的一员,IL-33可以与ILC2s表面表达的ST2-IL-1受体辅助蛋白复合体结合,活化ILC2s,并分泌大量Th2型细胞因子IL-5、IL-13等。IL-5可以促进嗜酸性粒细胞的聚集、加剧炎症反应;IL-13可以促进杯状细胞分泌黏液和平滑肌细胞收缩。初始CD4+T细胞可以分化为Th1或Th2细胞,Th1型细胞因子和Th2型细胞因子可以交叉调节Th2和Th1细胞的发育和分化。干扰素-γ(Interferon-γ,IFN-γ)可以由Th1细胞、CD8+T细胞、NK细胞和NKT细胞和其他细胞分泌,可以触发1型免疫反应、抑制2型免疫反应。然而IFN-γ对ILC2s功能的影响目前尚不清楚。鉴于ILC2s是Th2细胞的“镜像细胞”,我们猜测IFN-γ对ILC2s可能也存在抑制作用。研究目的本研究采用经鼻滴入IL-33的方法建立小鼠急性过敏性气道炎症模型,并用IFN-γ进行干预,观察IFN-γ对小鼠急性过敏性气道炎症的影响,并探究IFN-γ抑制小鼠急性过敏性气道炎症中ILC2s的作用机制及其对IL-5、IL-13产生的影响,为临床急性过敏性气道炎症的治疗提供理论依据。实验方法24只雌性C57BL/6(6-8w)小鼠随机分为4组,每组6只。IL-33模型组、单独IFN-γ组、PBS对照组、IL-33+IFN-γ联合刺激组。在实验第0 d和第3 d进行滴鼻刺激,第6 d处死小鼠收集支气管灌洗液(Bronchial alveolar lavage fluid,BALF)和肺组织。肺组织切片进行苏木素-伊红染色(Hematoxylin and eosin,HE)和过碘酸-雪弗染色(Periodic acid-Schiff reagent,PAS)观察病理变化。流式细胞术对ILC2s、嗜酸性粒细胞进行分析。酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)检测肺匀浆和BALF上清中IL-5和IL-13的含量。实时聚合酶链式反应(Real time polymerase chain reaction,Real-time PCR)检测IL-5、IL-13和ST2 m RNA表达量的变化。实验结果1.小鼠肺组织切片染色结果(1)HE结果显示:与PBS对照组相比,IL-33模型组细支气管及伴行血管周围有大量炎性细胞浸润;与IL-33模型组相比,IL-33+IFN-γ联合刺激组肺组织中仍有炎性细胞浸润,但明显减轻。(2)PAS结果显示:与PBS对照组相比,IL-33模型组支气管黏膜处可见大量杯状细胞增生且分泌大量黏液;与IL-33模型组相比,IL-33+IFN-γ联合刺激组支气管黏膜虽有杯状细胞增生和黏液分泌,但明显减少。2.小鼠BALF和肺单细胞悬液中ILC2s和嗜酸性粒细胞的流式结果流式结果显示:与PBS对照组相比,IL-33模型组BALF和肺组织中的嗜酸性粒细胞和ILC2s占总上样细胞数的比例和绝对值均升高(P0.05);与IL-33模型组相比,IL-33+IFN-γ联合刺激组BALF和肺组织中的嗜酸性粒细胞ILC2s占总上样细胞数的比例和绝对值均降低(P0.05)。3.小鼠BALF与肺匀浆上清中IL-5、IL-13 ELISA结果ELISA结果显示:与PBS对照组相比,IL-33模型组小鼠BALF和肺匀浆上清中的IL-5、IL-13含量升高(P0.05);与IL-33模型组相比,IL-33+IFN-γ联合刺激组BALF和肺匀浆上清中IL-5、IL-13含量降低(P0.05)。4.小鼠肺IL-5、IL-13、ST2 Real-time PCR结果Real-time PCR结果显示:与PBS对照组相比,IL-33模型组小鼠肺中IL-5、IL-13、ST2 m RNA表达量增加(P0.05);与IL-33模型组相比,IL-33+IFN-γ联合刺激组IL-5、IL-13、ST2 m RNA表达量降低(P0.05)。结论1.经鼻滴入IL-33可以成功诱导小鼠急性过敏性气道炎症模型。2.IFN-γ可以抑制ILC2s的增殖和活化并降低IL-5、IL-13的水平从而减弱炎症反应。
[Abstract]:Background allergic airway inflammation is the pathophysiological basis of allergic asthma, which is caused by an inappropriate immune response to external stimuli. It has long been thought that asthma is caused by the activation of antigen induced Th2 cells. Recent studies have shown that type 2 Group 2 innate lymphoid cells, ILC2s) in the process of asthma .ILC2s is widely distributed in fat related lymphoid clusters and non lymphoid tissues including skin, liver, lung, small intestine and visceral adipose tissue. Epithelial cell factor Thymic stromal lymphopoietin (TSLP), interleukin -33 (Interleukin-33, IL-33), IL-25 all can be found. Activated ILC2s.IL-33 is a member of the IL-1 family. IL-33 can combine with the ST2-IL-1 receptor auxiliary protein complex expressed on the surface of ILC2s, activate ILC2s, and secrete a large number of Th2 type cytokines, IL-5. IL-13 and other.IL-5 can promote the aggregation of eosinophils and aggravate the inflammatory reaction; IL-13 can promote the secreting mucus and smooth muscle fine of goblet cells. The initial CD4+T cells can differentiate into Th1 or Th2 cells. Th1 type cytokines and Th2 type cytokines can cross regulate the development and differentiation of Th2 and Th1 cells. Interferon gamma (Interferon- y, IFN- gamma) can be secreted by Th1 cells, CD8+T cells, NK cells and other cells, and can trigger type 1 immunoreaction and inhibit type 2 immunization. However, the effect of IFN- gamma on the function of ILC2s is not yet clear. Given that ILC2s is a "mirror cell" of Th2 cells, we suspect that IFN- gamma may also have a inhibitory effect on ILC2s. The purpose of this study was to establish an acute allergic airway inflammation model in mice by nasal drip into IL-33 and to observe the IFN- gamma with IFN- gamma intervention. The effect of IFN- on acute allergic airway inflammation in mice and the mechanism of ILC2s in acute allergic airway inflammation in IFN- gamma inhibition mice and its effect on the production of IL-5 and IL-13 were explored to provide a theoretical basis for the treatment of acute allergic airway inflammation. 24 female C57BL/6 (6-8W) mice were randomly divided into 4 groups with 6.IL-33 in each group. Model group, single IFN- gamma group, PBS control group, IL-33+IFN- gamma combined stimulation group. At zeroth D and third D, the nasal drip irritation was carried out, and sixth D were killed in the mice to collect bronchial lavage fluid (Bronchial alveolar lavage fluid, BALF) and lung tissue. Eriodic acid-Schiff reagent, PAS) observation of pathological changes. Flow cytometry was used to analyze ILC2s and eosinophils. Enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) was used to detect IL-5 and IL-13 in lung homogenate and BALF supernatant. CR) detection of changes in the expression of IL-5, IL-13 and ST2 m RNA. Experimental results 1. mice lung tissue section staining results (1) HE results showed: compared with the PBS control group, there was a large number of inflammatory cell infiltration around the bronchioles and the accompanying vessels in the IL-33 model group, and there was still inflammatory cell infiltration in the lung tissue of the IL-33+IFN- gamma stimulation group compared with the IL-33 model group. (2) PAS results showed that a large number of goblet cells proliferated and secreted a large number of mucus in the bronchial mucosa of the IL-33 model group compared with the PBS control group. Compared with the IL-33 model group, the bronchial mucosa of the IL-33+IFN- gamma combined stimulation group had goblet cell proliferation and mucus secretion, but significantly reduced the BALF and lung single cell suspension of.2. mice. The flow cytometric results of ILC2s and eosinophils showed that compared with the PBS control group, the proportion and absolute value of eosinophils and ILC2s in the BALF and lung tissues in the IL-33 model group were increased (P0.05), and BALF in the IL-33+IFN- gamma combined stimulation group and the eosinophil IL in the lung tissue compared with the IL-33 model group. The proportion and absolute value of the total number of C2s cells decreased (P0.05).3. mice BALF and lung homogenate supernatant, IL-5, IL-13 ELISA results ELISA results showed: compared with the PBS control group, IL-33 model group mice BALF and lung homogenate supernatant increased content; compared with the model group, the combined stimulation group and lung homogenization IL-5, IL-13 content decreased (P0.05).4. mice lung IL-5, IL-13, ST2 Real-time PCR results, and Real-time PCR results showed that compared with the PBS control group, the expression amount increased in the model group. Conclusion 1. IL-33 can successfully induce acute allergic airway inflammation in mice by nasal drip..2.IFN- gamma can inhibit the proliferation and activation of ILC2s and reduce the level of IL-5, IL-13 and thus weaken the inflammatory response.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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