CCL20-CCR6轴在呼吸道合胞病毒感染打破卵蛋白（OVA）口服免疫耐受小鼠模型中的作用

发布时间：2018-05-07 18:23

本文选题：呼吸道合胞病毒 + 巨噬细胞炎性蛋白20抗体　；参考：《皖南医学院》2017年硕士论文

【摘要】：目的:在前期实验研究中我们建立了RSV急性感染打破OVA口服耐受的小鼠模型,研究发现RSV急性感染能够打破OVA口服耐受使原本对OVA耐受的小鼠在雾化吸入OVA后肺部炎症显著加重,且CCL20在肺组织中表达增加,CCR6阳性的炎症细胞浸润明显增多。本研究将进一步研究CCL20-CCR6在RSV急性感染打破OVA口服耐受使肺部炎症加重的机制。方法:(1)在体外使用O、1、5、10 MOI的RSV感染(小鼠肺上皮细胞MLE-12系)MLE-12细胞,分别在6h、12h、24h三个不同时间点收集细胞样品,提取RNA,使用real time-PCR检测CCL20的mRNA表达水平;提取蛋白样品,应用免疫印迹法检测产生CCL20的信号通路中相关的蛋白表达,如:STAT3、IKKα及NF-κB。(2)在原有的RSV急性感染OVA口服耐受小鼠模型基础上给予腹腔注射CCL20中和抗体,以腹腔注射isotype IgG1抗体作为对照组,利用ELISA检测血清IgE,HE染色检测肺组织病理,RT-qPCR检测肺部炎症因子表达,肺泡灌洗液瑞氏染色检测炎症细胞,并用流式细胞术检测肺门淋巴结淋巴细胞类型,观察阻断CCL20-CCR6轴对RSV急性感染OVA口服耐受小鼠的影响。结果:(1)与control组比较,RSV感染MLE-12细胞后CCL20的mRNA的水平明显升高,且在12小时CCL20 mRNA的水平最高。选择12小时作为时间点比较不同MOI的RSV感染对MLE-12细胞CCL20 mRNA的水平的影响。发现在MOI=5时CCL20 mRNA的水平最高;随着时间的推移CCL20 mRNA水平随之降低。(2)与control组相比,在12小时以不同的MOI(1,5,10)的RSV感染MLE-12细胞,发现与调控CCL20表达的信号通路相关分子MyD88水平升高,STAT3、IKKα、NF-κB磷酸化水平升高;(3)在动物实验中,应用中和CCL20抗体阻断后,肺泡灌洗液中淋巴细胞明显减少,细胞总数、嗜酸性粒细胞、中性粒细胞均呈下降的趋势;血清IgE降低,肺组织炎症细胞浸润及气道上皮增生脱落明显改善;肺组织炎症因子IL-5、IL-13、Il-7A表达降低;肺门淋巴结CCR6+T淋巴细胞及CCR6+IL-17+细胞减少。结论:(1)RSV感染能够使上皮细胞CCL20表达增加,其机制可能是通过激活调控CCL20表达通路中的一些信号通路分子如:MyD88、STAT3、IKKα、NF-κB(2)阻断CCL20-CCR6轴能够减轻RSV急性感染OVA口服耐受小鼠的肺部炎症。
[Abstract]:Objective: to establish a mouse model of RSV acute infection to break the oral tolerance of OVA. It was found that acute RSV infection could break the oral OVA tolerance and increase the expression of CCL20 in lung tissue of OVA tolerant mice after atomization inhalation of OVA, and the infiltration of CCR6 positive inflammatory cells in lung tissue was significantly increased. This study will further investigate the mechanism of CCL20-CCR6 breaking OVA oral tolerance in acute RSV infection and exacerbating pulmonary inflammation. Methods the cell samples were collected at three different time points (6 h, 12 h and 24 h) to detect the mRNA expression level of CCL20 and the protein samples were extracted from MLE-12 cells of mouse lung epithelial cells infected with RSV for 10 MOI in vitro (MLE-12 cells of mouse lung epithelial cells), respectively, at three different time points (6 h, 12 h and 24 h), and the expression level of CCL20 was detected by real time-PCR. Immunoblotting was used to detect the expression of proteins related to the signaling pathway of CCL20 production, such as: STAT3IKK 伪 and NF- 魏 B.t2. The mice with acute OVA infection were given intraperitoneal injection of CCL20 neutralizing antibody on the basis of the original model of OVA oral tolerance induced by acute RSV infection. Isotype IgG1 antibody was injected intraperitoneally as control group. The expression of inflammatory factors in lung tissue was detected by ELISA staining with serum IgE and HE staining, and inflammatory cells were detected by RIA staining in alveolar lavage fluid. The lymphocyte types of hilar lymph nodes were detected by flow cytometry, and the effect of blocking CCL20-CCR6 axis on OVA oral tolerance mice with acute RSV infection was observed. Results compared with control group, the mRNA level of CCL20 in MLE-12 cells was significantly higher than that in control group, and the level of CCL20 mRNA was the highest at 12 hours. The effects of RSV infection with different MOI on the CCL20 mRNA level of MLE-12 cells were compared at 12 hours. It was found that the highest level of CCL20 mRNA was found at 5 CCL20 mRNA, and the CCL20 mRNA level decreased with time. Compared with control group, MLE-12 cells were infected with different RSV at 12 hours. It was found that the level of MyD88 associated with the signal pathway regulating the expression of CCL20 was increased. The phosphorylation level of STAT3IK 伪 -NF-kB was increased. In animal experiments, the lymphocytes in alveolar lavage fluid decreased, the total number of cells and eosinophilic granulocytes in alveolar lavage fluid were significantly decreased after the application of neutralizing and blocking of CCL20 antibody. The expression of IL-5, IL-13, Il-7A, CCR6 T lymphocytes and CCR6 IL-17 cells in hilar lymph nodes were decreased. Conclusion the expression of CCL20 in epithelial cells can be increased by CCL20 infection. The mechanism may be that blocking the CCL20-CCR6 axis by activating some signaling pathway molecules in the CCL20 expression pathway, such as: MyD88-STAT3KK 伪 -NF- 魏 B-2, can reduce the pulmonary inflammation in mice with acute RSV infection with OVA oral tolerance.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25;R-332

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