肺炎患者IL-17B水平升高及其介导支气管上皮细胞表达趋化因子IL-8的免疫机制研究

发布时间：2018-03-09 23:35

本文选题：肺部免疫　切入点：IL-17B　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:肺炎已成为危害人类健康的主要疾病之一。目前已有证据表明肺组织细胞在调控肺部免疫和感染中发挥着重要作用。支气管上皮细胞是抵御外部病原体的第一道防御屏障,肺成纤维细胞在肺结缔组织中含量丰富,是肺间质和血管的细胞通讯桥梁。肺部感染时,肺组织细胞通过分泌促炎细胞因子、生长因子、趋化因子以及相应的抗菌物质积极参与固有性和获得性免疫应答。研究肺组织细胞在肺部免疫和感染的作用,有助于预测、预防和治疗肺炎及其他呼吸道疾病。IL-17B作为IL-17家族中的一员,虽在免疫性胃肠道疾病中有所提及,但其在肺部免疫中的作用尚未有报道。因此,研究肺部感染中肺组织细胞如何对IL-17B产生免疫应答以及通过何种分子机制调控该途径,有助于我们了解肺部感染免疫的进程,为临床治疗提供策略。方法:收集社区获得性肺炎(CAP)患者和正常血液标本(成人肺炎患者/正常成人=37/14,儿童肺炎患者/正常儿童=28/14),ELISA检测两组标本IL-17B和IL-8水平差异及相关性;Q-PCR检测IL-17B处理人支气管上皮细胞(BEAS-2B)和人肺成纤维细胞(HLF)后,相关细胞因子m RNA的表达差异;TNF-α、IFN-γ单独或与IL-17B联合处理人支气管上皮细胞24小时后,ELISA检测细胞培养上清中IL-8水平的变化;用信号通路抑制剂处理人支气管上皮细胞1小时后,再用IL-17B处理细胞24小时,ELISA检测细胞上清IL-8水平变化;IL-17B处理BEAS-2B不同时间点,western blot检测细胞内信号通路蛋白的磷酸化水平;分别用铜绿假单胞菌、金黄色葡萄球菌、大肠杆菌和肺炎链球菌构建小鼠肺炎模型,ELISA检测小鼠支气管肺泡灌洗液和血清中IL-17B和IL-8的水平。结果:CAP患者血清中IL-17B和IL-8水平显著升高且两者呈正相关。IL-17B可诱导人支气管上皮细胞表达IL-8显著升高,且具有一定的剂量依赖性。TNF-α可显著增强IL-17B诱导BEAS-2B表达IL-8,而IFN-γ则显著降低IL-17B刺激BEAS-2B产生IL-8。IL-17B刺激人支气管上皮细胞5分钟后,NF-κB、Akt、ERK、P38 MAPK的磷酸化水平显著升高;采用NF-κB抑制剂、Akt抑制剂、ERK抑制剂、P38MAPK抑制剂联合IL-17B处理BEAS-2B组较IL-17B单独处理组的IL-8水平显著降低,且NF-κB、Akt、ERK、P38 MAPK磷酸化水平显著减弱。不同细菌构建的小鼠肺炎模型中,IL-17B和IL-8的水平都显著升高且两者正相关。结论:肺部感染时,IL-17B通过激活人支气管上皮细胞NF-κB、Akt、ERK、P38 MAPK信号通路,上调IL-8的表达水平,进一步促进免疫应答。
[Abstract]:Objective: pneumonia has become one of the major diseases endangering human health. At present, there is evidence that lung tissue cells play an important role in regulating lung immunity and infection. Bronchial epithelial cells are resistant to external pathogens. The first defense barrier, Lung fibroblasts are abundant in the connective tissue of the lung and act as a bridge between the interstitial cells and the blood vessels of the lung. In lung infection, lung tissue cells secrete inflammatory cytokines, growth factors, and growth factors. Chemokines and corresponding antimicrobial substances are actively involved in innate and acquired immune responses. Studying the role of lung tissue cells in lung immunity and infection helps to predict, Prevention and treatment of pneumonia and other respiratory diseases. IL-17B, as a member of the IL-17 family, has been mentioned in immune gastrointestinal diseases, but its role in lung immunity has not been reported. It is helpful to understand the immune process of lung infection by studying the immune response of lung tissue cells to IL-17B and the molecular mechanism through which this pathway is regulated. Methods: IL-17B and IL-8 levels in patients with community-acquired pneumonia (CPAP) and normal blood samples (adult pneumonia / normal adults 37 / 14, pediatric pneumonia / normal children 28 / 14) were detected by Elisa. And the Q-PCR method was used to detect the changes of human bronchial epithelial cells (BEAS-2B) and human lung fibroblasts (HLF) treated with IL-17B. The difference of expression of related cytokine m RNA and the changes of IL-8 level in the supernatant of human bronchial epithelial cells treated with TNF- 伪 -IFN- 纬 alone or in combination with IL-17B for 24 hours were detected by Elisa, and the level of IL-8 in the supernatant of human bronchial epithelial cells treated with signal pathway inhibitor for 1 hour. The changes of IL-8 level in supernatant of cells treated with IL-17B for 24 hours were assayed by Elisa. The phosphorylation level of intracellular signal pathway proteins was detected by western blot at different time points of BEAS-2B treated with IL-17B, Pseudomonas aeruginosa and Staphylococcus aureus were used respectively. Elisa was used to detect the levels of IL-17B and IL-8 in bronchoalveolar lavage fluid and serum of mice induced by E. coli and Streptococcus pneumoniae. The expression of IL-8 in human bronchial epithelial cells was significantly increased. In a dose-dependent manner, TNF- 伪 could significantly enhance the expression of IL-8 in BEAS-2B induced by IL-17B, while IFN- 纬 significantly decreased the phosphorylation level of P38 MAPK in human bronchial epithelial cells stimulated by BEAS-2B and IL-8.IL-17B stimulated by IL-17B for 5 minutes. The level of IL-8 in the BEAS-2B group treated with NF- 魏 B inhibitor P38 MAPK and IL-17B was significantly lower than that in the IL-17B alone group. Moreover, the phosphorylation level of NF- 魏 B and AktERKN P38 MAPK was significantly decreased. The levels of IL-17B and IL-8 were significantly increased in mice with pneumonia induced by different bacteria. Conclusion: during pulmonary infection, IL-17B activates the P38 MAPK signaling pathway in human bronchial epithelial cells (NF- 魏 B). Upregulate the expression of IL-8 and further promote the immune response.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.1

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