發(fā)布時間：2018-05-07 18:23

  本文選題：呼吸道合胞病毒 + 巨噬細胞炎性蛋白20抗體��； 參考：《皖南醫(yī)學院》2017年碩士論文

【摘要】：目的:在前期實驗研究中我們建立了RSV急性感染打破OVA口服耐受的小鼠模型,研究發(fā)現(xiàn)RSV急性感染能夠打破OVA口服耐受使原本對OVA耐受的小鼠在霧化吸入OVA后肺部炎癥顯著加重,且CCL20在肺組織中表達增加,CCR6陽性的炎癥細胞浸潤明顯增多。本研究將進一步研究CCL20-CCR6在RSV急性感染打破OVA口服耐受使肺部炎癥加重的機制。方法:(1)在體外使用O、1、5、10 MOI的RSV感染(小鼠肺上皮細胞MLE-12系)MLE-12細胞,分別在6h、12h、24h三個不同時間點收集細胞樣品,提取RNA,使用real time-PCR檢測CCL20的mRNA表達水平;提取蛋白樣品,應用免疫印跡法檢測產生CCL20的信號通路中相關的蛋白表達,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礎上給予腹腔注射CCL20中和抗體,以腹腔注射isotype IgG1抗體作為對照組,利用ELISA檢測血清IgE,HE染色檢測肺組織病理,RT-qPCR檢測肺部炎癥因子表達,肺泡灌洗液瑞氏染色檢測炎癥細胞,并用流式細胞術檢測肺門淋巴結淋巴細胞類型,觀察阻斷CCL20-CCR6軸對RSV急性感染OVA口服耐受小鼠的影響。結果:(1)與control組比較,RSV感染MLE-12細胞后CCL20的mRNA的水平明顯升高,且在12小時CCL20 mRNA的水平最高。選擇12小時作為時間點比較不同MOI的RSV感染對MLE-12細胞CCL20 mRNA的水平的影響。發(fā)現(xiàn)在MOI=5時CCL20 mRNA的水平最高;隨著時間的推移CCL20 mRNA水平隨之降低。(2)與control組相比,在12小時以不同的MOI(1,5,10)的RSV感染MLE-12細胞,發(fā)現(xiàn)與調控CCL20表達的信號通路相關分子MyD88水平升高,STAT3、IKKα、NF-κB磷酸化水平升高;(3)在動物實驗中,應用中和CCL20抗體阻斷后,肺泡灌洗液中淋巴細胞明顯減少,細胞總數(shù)、嗜酸性粒細胞、中性粒細胞均呈下降的趨勢;血清IgE降低,肺組織炎癥細胞浸潤及氣道上皮增生脫落明顯改善;肺組織炎癥因子IL-5、IL-13、Il-7A表達降低;肺門淋巴結CCR6+T淋巴細胞及CCR6+IL-17+細胞減少。結論:(1)RSV感染能夠使上皮細胞CCL20表達增加,其機制可能是通過激活調控CCL20表達通路中的一些信號通路分子如:MyD88、STAT3、IKKα、NF-κB(2)阻斷CCL20-CCR6軸能夠減輕RSV急性感染OVA口服耐受小鼠的肺部炎癥。
[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 偽 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 偽 -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 偽 -NF- 魏 B-2, can reduce the pulmonary inflammation in mice with acute RSV infection with OVA oral tolerance.
【學位授予單位】：皖南醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R562.25;R-332

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