【作者】 Binaya Wasti；

【导师】 向旭东；

【作者基本信息】 中南大学 ， 呼吸内科， 2010， 硕士

【摘要】 PPARγ激动剂下调急性哮喘CD4+T淋巴细胞IL-17的表达背景：哮喘是一种最常见的炎症性疾病,在近二十年其发病率顯著增加.嗜酸细胞,巨噬细胞,T淋巴细胞,肥大细胞和上皮细胞参与炎症过程。最近认为,Th 17细胞和它的细胞因子(IL-17)发病的病理生理过程。PPARγ已经被证实在急性哮喘的呼吸道炎症反应的控制中具有重要作用。目标：1)确认由卵白蛋白致敏,复制小鼠急性哮喘动物模型。2)评估PPARγ激动剂对从急性哮喘小鼠CD4+T淋巴细胞IL-17表达的作用并与地塞米松对比。方法：将小鼠分为对照组(正常)组和哮喘组。急性哮喘小鼠用卵白蛋白致敏及经气道激发,对照组用PBS。急性哮喘小鼠模型的确认是通过评估行为变化,哮喘症状,用乙酰甲胆碱激发行肺功能测试,BALF的炎症细胞,IL-17水平变化和肺组织病理。第21天,处死所有的小鼠,取出脾脏。用免疫磁珠法分离,纯化对照组和哮喘组脾来源的CD4+T淋巴细胞,用台盼蓝染色判断其活力；测定。CD4+T淋巴细胞中的Th-17细胞用流式细胞仪检测。用ELISA方法测定哮喘组及正常组培养24小时上清液IL-17的浓度；哮喘组再分为哮喘对照组(Acon),罗格列酮组(Aros)和地塞米松(DXM)组(Adex)。分别加入罗格列酮和DXM培养24小时后,而正常对照组CD4+T淋巴细胞为未干预(Con),以与Acon对比；分别用免疫印迹(WB)和实时聚合酶链反应(real-time PCR)检测所有组的IL-17蛋白和IL-17mRNA的表达.结果：Ⅰ确认急性哮喘模型1.卵白蛋白致敏和雾化后的小鼠的变化卵白蛋白致敏组小鼠最先表现出兴奋,抓耳,打喷嚏,经气道激发后出现嗜睡,少动,进食减少。对照(正常)组小鼠用PBS致敏和雾化后,灵活度、活动、行为和饮食习惯均正常。2.BALF检测哮喘组总细胞计数,中性粒细胞,嗜酸性粒细胞,巨噬细胞和淋巴细胞水平及IL-17水平与正常组比较,均上升,均P<0.01。3.肺功能测试与正常对照组相比,随着乙酰甲胆碱浓度的增加,哮喘组肺和气道阻力逐渐增加,均P<0.01。4.肺组织学结果哮喘组的炎症细胞,如嗜酸性粒细胞、中性粒细胞、巨噬细胞较正常组比较,均显著上升,P<0.01.Ⅱ正常组和哮喘组CD4+T淋巴细胞的变化经过24小时的培养,哮喘组CD4+T淋巴细胞浓度(4.164±0.260 X1010／L)高于正常组(1.138±0.130 X 10m／L),P<0.01.Ⅲ培养24小时后,正常组和哮喘组CD4+T淋巴细胞中Th 17细胞的变化哮喘组Th 17细胞高于正常组,P<0.01.Ⅳ正常组和哮喘组CD4+T淋巴细胞细胞培养上清液中IL-17的变化哮喘组CD4T淋巴细胞细胞培养上清液中IL-17高于正常组,P<0.01.V培养CD4+T-淋巴细胞IL-17蛋白和mRNA的变化及罗格列酮和DXM的影响1.免疫印迹法检测各组CD4+T-淋巴细胞IL-17蛋白的表达哮喘对照组IL-17浓度较正常组明显增加,p<0.01。罗格列酮组和DXM组中CD4+T淋巴细胞中IL-17的水平低于哮喘对照组,均P<0.01,但罗格列酮组和DXM组间CD4+T淋巴细胞中IL-17的表达无差异,均P>0.05。2.实时PCR检测各组CD4+T-淋巴细胞IL-17 mRNA的表达各组中哮喘对照组IL-17 mRNA表达水平最高,P<0.01,罗格列酮组和DXM组中CD4+T淋巴细胞中IL-17mRNA的水平低于哮喘对照组,均P<0.01,但罗格列酮组和DXM组间CD4+T淋巴细胞中IL-17 mRNA的水平无差异,P>0.05。结论：1.成功建立急性哮喘小鼠动物模型。2.IL-17参与了急性哮喘中性粒细胞气道炎症过程。3. PPARγ激动剂罗格列酮能下调急性哮喘CD4+T淋巴细胞中IL-17的表达。4.DXM下调急性哮喘CD4+T淋巴细胞中IL-17的表达。更多还原

【Abstract】 Peroxisome Proliferator-Activated Receptorγ(PPARγ) agonist down regulates IL-17 expression from CD4+ T-lymphocytes in acute asthmaBackground:Asthma is one of the most common inflammatory diseases and its incidence is dramatically increasing from the last twenty years.Eosinophils,Macrophages,T-lymphocytes, mast cells and epithelial cells are involved in the process of inflammation. Recently it has been suggested that Th 17 and its cytokines(IL-17)are involved in the pathogenesis of asthma in mouse model.PPARγhas been shown to play an important role in the control of inflammatory responses of airways in acute asthma.Objectives:1)To confirm the Ovalbumin sensitized and airway challenged acute asthma model.2) To evaluate the down regulating effect of PPARγagonist on IL 1.7 expression isolated from CD4+ T-lymphocytes from the spleen of acute asthmatic mice.Methods:Mice were divided into control (normal)group and acute asthmatic group. We used mouse model of acute asthma induced by sensitization and airway challenge with ovalbumin and only by PBS in control (normal)group. The acute asthma model was confirmed by evaluating the behavioral changes and asthmatic symptoms occurred in mice and through the pulmonary function test with methacholine provocation, level of inflammatory cells and IL-17 in BALF and lung tissue histology. On day 21st,all the mice were sacrificed and spleen was taken out.CD4+ T-lymphocytes from the spleen of control and asthmatic groups were isolated,purified by immuno-magnetic beads method.The CD4+ T-lymphocytes viability was assessed by trypan blue staining and the level of Th-17 cells in CD4+ T-lymphocytes assessed by flow cytometry. The level of IL-17 in CD4+ T-lymphocytes culture supernatant was measured by ELISA. The correlation between BALF IL-17 concentration and BALF neutrophil(%)was assessed. The isolated CD4+ T-lymphocyte were divided into Nontrol group (Con),Asthmatic control group (Acon),Asthmatic rosiglitazone group (Aros)and Asthmatic Dexamethasone group (Adex).After the addition of rosiglitazone and dexamethasone, and then these cells were cultured for 24 hours,IL-17 protein and IL-17 mRNA of CD4+ T-lymphocytes from all the groups were detected by Western Blot(WB)and real-time Polymerase Chain Reaction(rt-PCR)respectively.Results:I Confirmation of acute asthma model1 Changes in mice after sensitization and nebulization with OVAOVA sensitized group mice showed excitation, ear grasping, sneezing at the beginning and drowsiness, lack of activities and reduced eating habit later after subsequent airway challenge with OVA. Control (Normal)group mice sensitized and nebulized only with PBS showed normal sensitivity, activity, behaviour and eating habit.2 BALF examinationTotal cellular counts,neutrophils,eosinophils,macrophages and lymphocytes level increased in acute asthmatic group as compared to normal group, all P<0.01 respectively.BALF IL-17 level increased in acute asthmatic group as compared to normal group, p<0.01.3 Pulmonary Function Test (PFT) interpretationWith the subsequent increase in methacholine doses, the progressive increase in lung and airway resistance recorded in acute asthmatic group as compared to normal group, all p <0.01 respectively.4 Lung histology Acute asthmatic group showed significant increase in inflammatory cells like eosinophils,neutrophils macrophages as compared to normal group, p<0.01.ⅡThe level of CD4+ T-lymphocytes in normal and asthmatic group After 24 hours of culture, the acute asthmatic group showed higher concentration of CD4+ T-lymphocytes than the normal group,(4.164±0.260 X 1010/L) vs(1.138±0.130 X 10/L,p <0.01.III Level of Th-17 cells in CD4+ T-lymphocytes in normal and asthmatic group after 24 hours of culture The acute asthmatic group showed higher level of Th 17 cells than the normal group, p<0.01.IV Level of IL-17 in CD4+ T lymphocytes culture supernatant after 24 hours CD4+ T-lymphocytes derived from the acute asthmatic mice showed obvious increase in IL-17 concentration than the normal group, p<0.01.V Impact of Rosiglitazone and Dexamethasone in IL-17 protein and mRNA expression on cultured CD4+T-lymphocytes1)Expression of IL-17 protein from CD4+ T-lymphocytes in all groups detected by Western BlotAsthmatic group(Acon)showed increased level of IL-17 protein expression than that of other groups, p<0.01 respectively. The Aros and Adex group showed decreased IL-17 protein expression from CD4+ T-lymphocytes than the Acon group, all p<0.01.But the Aros and Adex group showed no difference in IL-17 mRNA expression from CD4+ T-lymphocytes,p>0.05. 2)Expression of IL-17 mRNA from CD4+ T-lymphocytes in all groups detected by real-time PCR Asthmatic group(Acon)showed increased level of IL-17 mRNA expression than that of other groups,p<0.01 respectively. The Aros and Adex group showed decreased IL-17 mRNA expression from CD4+ T-lymphocytes than the Acon group, all p<0.01.But the Aros and Adex group showed no any differences in IL-17 mRNA expression from CD4+ T-lymphocytes, p>0.05.Conclusion:1.Acute asthma model was successfully established.2.IL-17 was involved in neutrophilic airway inflammation in acute asthma.3.PPAR y agonist down regulates the IL-17 expression from CD4+ T-lymphocytes in acute asthma.4. Dexamethasone also found to down regulate the IL-17 expression from CD4’T-lymphocytes in acute asthma.更多还原