【作者】 张清玲；

【导师】 钟南山； 赖克方；

【作者基本信息】 广州医学院 ， 呼吸内科学， 2008， 博士

【摘要】 研究背景和目的:嗜酸粒细胞性支气管炎(eosinophilic bronchitis,EB)是慢性咳嗽的主要原因。诱导痰和气道粘膜病理检查表明,EB同样存在以嗜酸性粒细胞为主的多种炎症细胞浸润。但EB为何缺乏气道高反应性,EB是否会发展为气道高反应性或支气管哮喘(bronchial asthma)?本研究通过观察EB的临床特征及免疫病理特征,分析其与支气管哮喘患者的差异。同时,通过研究EB和哮喘支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)细胞蛋白质组双向电泳表达谱,找出表达差异蛋白,为筛选气道高反应性和气流可逆性阻塞相关的特异蛋白、探讨哮喘气道高反应性和气流可逆性的发病机制奠定基础。在此基础上,探讨建立缺乏气道高反应性的EB动物模型的可能性和途径,试图从动物实验进一步深入研究EB和哮喘的关系,为支气管哮喘的早期干预寻找新的靶点。结果及结论:1. EB和哮喘的气道炎症病理特点相似,涉及多种炎症细胞,包括嗜酸性粒细胞(eosinophil,Eos)、T淋巴细胞和肥大细胞等,但EB的气道炎症程度较哮喘轻,炎症范围更为局限。2.双向电泳图像对比分析表明,EB与哮喘之间BLAF细胞蛋白表达存在一定的差异。本研究成功鉴定了EB、哮喘之间的2个差异表达蛋白,其中谷胱甘肽-S-转移酶T1(glutathione-S-transferase T1,GSTT1)可能与气道反应性的发生机制密切相关。3.初步成功建立了具有明显的Eos气道炎症、但无气道高反应性的BALB/c小鼠EB模型,为进一步了解EB的气道炎症及气道高反应性的发生机制奠定了基础。第一部分:嗜酸粒细胞性支气管炎与支气管哮喘患者免疫病理特征的比较目的:了解EB气道的病理学特征,比较其与支气管哮喘气道炎症的异同。方法:对20例EB、24例支气管哮喘和10例正常人进行诱导痰检查,分析诱导痰的细胞分类;经支气管纤维镜支气管肺泡灌洗(bronchoalveolar lavage,BAL),分析BALF的细胞分类;于隆突、左舌叶开口、右下叶前段取支气管粘膜活检,光镜下观察活检标本的病理结构特征,测量气道上皮粘膜的基底膜厚度,通过免疫组化技术,观察气道粘膜上皮层和上皮下层炎症细胞(Eos、肥大细胞、T淋巴细胞)的分布情况并计算其细胞分布密度。实验结果中的计量资料用中位数表示,组间比较采用非参数秩和检验。结果:1. EB组痰Eos百分率[10.13%(20.19)]显著低于哮喘组[26.00%(32.97)](P<0.05)。EB组和哮喘组诱导痰Eos百分率均显著高于对照组[0.25%(0.50)](P<0.001)。2. EB组BALF的Eos百分率[1.63%(3.81)]显著低于哮喘组[11.75%(15.70)](P<0.001),EB组和哮喘组BALF的Eos百分率均显著高于对照组[0.38%(1.00)(P<0.05)。3.哮喘组舌叶开口支气管粘膜基底膜厚度[4.73μm(2.61)]显著大于正常对照组[3.23μm(1.35)](P<0.001)和EB组[3.41μm(1.89)](P<0.01),但EB组和正常对照组之间无差异(P>0.05)。哮喘组右下叶前段支气管粘膜基底膜厚度[5.36μm(3.65)]显著大于EB组[3.53μm(3.18)](P<0.05)。)4.比较舌叶支气管粘膜上皮下层的Eos分布密度,哮喘组[8.91个/mm2 (52.88)]显著高于EB组[0.00个/mm2 (10.40)]和正常对照组[0.00个/mm2 (0.00)](P<0.05)。5.比较舌叶支气管粘膜上皮层的淋巴细胞分布密度,EB组[475.76个/mm2(244.75)]和哮喘组[583.29个/mm2(507.69)]显著高于正常对照组[260.71个/mm2(430.31)] ,而EB和哮喘组差异无显著性(P>0.05)。6.比较舌叶支气管粘膜的肥大细胞分布密度:在粘膜上皮层,EB组[168.80个/mm(2314.28)]和哮喘组[252.78个/mm(2545.68)]显著高于正常对照组[0.00个/mm2(13.61)];在粘膜上皮下层,EB组[218.44个/mm2(383.65)]和哮喘组[197.22个/mm2(208.54)]显著高于正常对照组[66.07个/mm2(45.92)];而EB和哮喘组差异无显著性(P>0.05)。结论:EB和哮喘的气道炎症病理特点相似,涉及嗜酸性粒细胞、T淋巴细胞和肥大细胞等多种炎症细胞,但EB的气道炎症程度较哮喘轻,炎症范围更为局限。第二部分:嗜酸粒细胞性支气管炎与支气管哮喘分子生物基础差异的初步探讨研究背景和目的:在前面的研究中,我们发现EB和哮喘气道炎症病理尽管有很多相似的地方,但也有明显的差异。支气管肺泡灌洗液能够比较直接和客观地反映气道炎症的变化,被认为是蛋白组学研究气道炎症性疾病的理想样本。本实验的目的是利用比较蛋白质组学方法,探讨EB与支气管哮喘差异的分子生物学基础,以筛选鉴定与气道高反应性、气流可逆性阻塞相关的特异蛋白。方法:收集EB 5例、哮喘和正常人各6例,经纤维支气管镜于右中叶行支气管肺泡灌洗取得BALF,提取BALF细胞总蛋白质后,利用固相pH梯度(immobilized pH gradient, IPG),双向凝胶电泳(two-dimensional electrophoresis, 2-DE)对总蛋白进行分离,然后行硝酸银染色,予ImageMaster 2D Elite 5.0分析软件进行凝胶图像分析,寻找EB、哮喘和正常对照组差异表达的蛋白质。应用基质辅助电离解析飞行时间质谱(matrix-assitsted laser desorption / ionization time–of -flight mass spectrometry, MALDI-TOF-MS)获得蛋白质的肽质量指纹图谱(peptide mass fingerprint, PMF),再通过MALDI-TOF/TOF-MS串级质谱对蛋白质进行序列分析,得到蛋白质二级质谱图。最后利用Mascot软件搜索NCBInr蛋白质数据库,将获得的肽段信息进行对比分析,明确是何种蛋白质。结果:双向电泳图像对比分析表明,EB、哮喘和正常对照组BLAF细胞蛋白表达有一定的差异。各蛋白斑点经质谱检测肽质量指纹谱与标准分子量、等电点对照分析鉴定出2个蛋白,分别为铁蛋白(ferritin, light polypeptide)和谷胱甘肽-S-转移酶T1(glutathione-S-transferase T1,GSTT1),其中铁蛋白在哮喘组表达上调。EB组中GSTT1高表达,而83.3%哮喘患者和33.3正常对照不表达GSTT1。结论:1.电泳图像对比分析表明,EB与哮喘之间BLAF细胞蛋白表达存在一定的差异。2.成功鉴定了EB和哮喘之间的2个差异表达蛋白,其中GSTT1可能与气道反应性的发生机制密切相关。第三部分嗜酸粒细胞性支气管炎动物模型的建立及其与哮喘动物模型的气道炎症的比较分题1小鼠气道反应性方法的建立目的:建立过敏性小鼠气道反应性检查的正确方法。方法:采用SPF级BALB/c雌性小鼠,通过OVA致敏和激发的方法建立过敏性气道炎症模型(下称OVA组,n=36),第0、7、14d分别给予10μg OVA+1.3mg Al(OH)3生理盐水混悬液200μl腹腔注射致敏,第28d予100ug OVA滴鼻激发。生理盐水作为正常对照(下称NS组,n=36)。根据肺功能检查方法的不同再细分为4组:无创OVA组,无创NS组,有创OVA组,有创NS组,每组18只。气道反应性检查采用Buxco公司有创肺功能检查系统(“RC”系统)及无创肺功能检查系统(“Penh”系统)。激发24小时(第29天)后,以吸入倍增浓度的乙酰甲胆碱(methacholine ,Mch)雾化液[浓度依次为0.39、0.78、1.56、3.12、6.25、12.5、25、50(mg/ml)检查小鼠的气道反应性。有创检查时,以肺阻力(lung resistance,RL)为观察指标,通过药物的剂量-反应曲线来评价动物的气道反应性。无创检查时,以Penh为观察指标,通过药物的剂量-反应曲线及Penh较基础值上升100%、200%的吸入Mch的浓度(Penh200、Penh300)来评价动物的气道反应性。检查BALF细胞学分类和上气道、肺组织病理改变。另予0.3mM,3.0mM和30mM组织胺对OVA和NS组进行鼻组胺激发试验,每次激发后,观察10分钟内小鼠的打喷嚏和擦鼻次数,通过药物的剂量-反应曲线比较两组的差异。结果:1.OVA组及NS组Penh%,RL%均有随Mch浓度升高而增加的趋势,吸入浓度为6.25、12.5、25mg/ml的Mch时,无创OVA组Penh%显著高于无创NS组(P<0.05 ),且Penh200、Penh300显著低于无创NS组(P<0.001)。而有创OVA组气道反应性与有创NS组无显著差异(P>0.05 )。2.OVA组BALF中性粒细胞、嗜酸性粒细胞百分率显著高于NS组(P<0.01)。与NS组比较,OVA组肺组织炎症改变明显(P<0.01)。而无创OVA组BALF的Eos百分比[(27.06±12.05)%]和细胞总数[(2.69±0.55)×105/ml)]与有创OVA组[(25.33±10.79)%,(2.86±0.55)×105/ml)]无显著差异,两组下呼吸道组织病理评分亦无显著差异(P>0.05 )。3.鼻组胺激发试验显示,在30mM组织胺浓度时,OVA组擦鼻次数[(73.00±28.30)次]显著高于对照组[(43.40±34.07)次](P<0.05 )。OVA组上呼吸道病理检查显示上呼吸道轻度Eos炎症改变。结论:1.无创检查法较有创检查法对同一气道过敏疾病模型气道反应性的结果不相一致,其原因可能是模型气道炎症程度较轻,无创检查法受到上呼吸道阻力增加的影响所致。2.首次检查小鼠过敏性气道炎症模型气道反应性时,建议先用有创检查方法进行确定。分题2嗜酸粒细胞性气道炎症的小鼠模型的建立目的:通过调节抗原激发的时间、途径和剂量,建立无气道高反应性的BALB/c小鼠EB模型。方法:56只BALB/c雌性小鼠,分为4组:气道过敏雾化组(下称OVA-雾化组,n=8)、气道过敏1次滴鼻组(下称OVA 1次滴鼻组,n=12)、气道过敏2次滴鼻组(下称OVA 2次滴鼻组,n=8)、生理盐水对照组(下称对照组, n=28)。致敏方法同前。OVA-雾化组第28d予1%OVA雾化30分钟激发;OVA-1次滴鼻组第28天予100ug OVA滴鼻激发;OVA-2次滴鼻组第28d,第29天各予100ug OVA滴鼻激发。对照组因各模型组致敏、激发时间采用生理盐水进行致敏和激发。最后一次激发24小时后,有创检查法测定动物的气道反应性,并观察气道炎症情况。结果:1.气道反应性的测定:激发24小时后,各组RL%均有随Mch浓度升高而增加的趋势。在吸入浓度为0.39-50mg/ml的Mch时,OVA 1次滴鼻组和OVA雾化组RL%较对照组无显著性差异(P > 0.05);在吸入浓度为25mg/ml的Mch时,OVA 2次滴鼻组RL%显著高于对照组(P < 0.05)。2.各模型组巨噬细胞百分率显著低于对照组(P<0.05),而中性粒细胞、嗜酸性粒细胞百分率和淋巴细胞百分率显著高于对照组(P<0.05)。3. OVA雾化组巨噬细胞百分率显著高于OVA1次滴鼻组和OVA 2次滴鼻组(P<0.01,P<0.001),而嗜酸性粒细胞百分率[(7.40±3.41)%]显著低于OVA 1次滴鼻组[(25.33±10.79)%]和OVA 2次滴鼻组[(39.73±6.02)%](P<0.01,P<0.001)。结论:通过调节抗原激发的时间、途径和剂量,本实验初步成功建立了具有明显的Eos气道炎症、但无气道高反应性的BALB/c小鼠EB模型,为进一步研究EB的气道炎症及气道高反应性的发生机制奠定了基础。分题3嗜酸粒细胞性支气管炎动物模型的气道炎症及其与哮喘动物模型的比较目的:观察BALB/c小鼠EB模型的气道炎症特征并比较其与哮喘动物模型的气道炎症的差异。方法:80只BALB/c雌性小鼠,分为3组:EB模型组(下称EB组,n=28)、哮喘模型组(下称哮喘组,n=12)、生理盐水对照组(下称对照组,n=40)。致敏方法同前。EB模型组第28d予100ug OVA滴鼻激发;哮喘组第28,29,30d予100ug OVA滴鼻激发(每天1次)。对照组因每组致敏、激发时间采用生理盐水进行致敏和激发。在末次激发24小时后,各组(n=12)分别检测小鼠的下列指标:有创检查法测定气道高反应性,支气管肺泡灌洗液(BALF)细胞总数及细胞分类,支气管及肺组织炎症程度。此外,在末次激发12、48小时后,分别观察EB组和对照组气道高反应性、BALF细胞分类的变化情况(每组n=8)。结果:1.气道反应性的测定:激发12小时、24小时及48小时后,EB组气道反应性与对照组均无显著差异(P>0.05 )。而哮喘组在末次激发24小时后,在吸入浓度为12.5、25、50mg/ml的Mch时,RL%均显著高于对照组(P<0.05)。2.气道炎症:EB组滴鼻激发12小时、24小时、48小时后BALF的Eos百分比呈逐渐增加的趋势(P<0.05)。激发24小时后,EB、哮喘模型组BALF巨噬细胞百分率显著低于对照组(P<0.05),而中性粒细胞、嗜酸性粒细胞百分率和淋巴细胞百分率显著高于对照组(P<0.05)。哮喘组BALF的Eos百分比[(42.15±13.35)%]和细胞总数[(7.59±1.33)×105/ml)]明显高于EB组[(25.33±10.79)%,(2.86±0.55)×105/ml)] (P<0.05),而中性粒细胞百分比[(11.14±6.61)%]显著低于EB组[(23.89±23.67)%](P<0.05)。激发24小时后,哮喘组支气管肺部炎症组织病理评分显著高于EB组(P<0.05)。结论:1.随着激发后时间的增加,EB模型的Eos气道炎症有逐步加重趋势,但并未观察到气道反应性的变化。2.本研究建立的EB模型的气道炎症程度轻于哮喘模型组。更多还原

【Abstract】 Background and ObjectiveEosinophilic bronchitis (EB) is an important etiological factor inducing chronic cough. The analysis of induced sputum and bronchial mucosal biopsies from EB patients showed that the airway inflammation was similar to that seen in asthma. However, it remains open to question why no functional abnormalities associated with asthma was observed in EB patients and what its underlying mechanisms are contributing to the pathophysiology of asthma. In the present study, we investigate the airway pathological characteristics of EB and use the comparative proteomics technique to explore at the molecular level differences between eosinophilic bronchitis and bronchial asthma for identifying idiotype proteins associated with the airway hyperresponsiveness and the reversible obstruction. Furthermore, we try to establish an eosinophilic bronchitis mouse model without airway hyperresponsiveness based on our clinical work.Results and conclusion1. The pathological features of the airway inflammation in EB and asthma are similar. It is an airway inflammatory disorder characterized by infiltration of various inflammation cells (eosinophils, T lymphocytes and mast cells). However, the intensity of airway inflammation in EB is milder, and the extent is more limited than that in asthma.2. By comparative proteomics technique, we have succeeded in the identification of two proteins differentially expressed in the EB, asthma and normal group, and we found that GSTT1 might be closely related to the development of airway responsiveness.3. We established a preliminary EB animal model successfully. The intensity of airway inflammation in this EB model was milder than that in the asthma model. Our model established a foundation for the further research.PartⅠComparison in Immunopathology between Eosinophilic Bronchitis and AsthmaObjectiveTo investigate the airway pathological characteristics of eosinophilic bronchitis (EB) in comparison with asthmaMethodsInduced sputum analysis was performed in 20 cases with EB, 24 cases with bronchial asthma and 10 cases of healthy subjects. Differential cell counts in induced sputum, fiber optic bronchoscopy, and bronchoalveolar lavage (BAL) were performed, followed by cell counting, and classification. The biopsy specimens of bronchial mucosa were taken from the carinae, the gap between the left lingual lobe and the anterior segment (B8) of the right and lower lung. The biopsy specimens of the control group were just taken from the gap of lingual lobe. Pathological features of the biopsy specimens were observed by Light microscopy, and the thickness of the basement membrane of the epithelial mucosa was measured. Using immunohistochemical analysis, the distribution of inflammatory cells (Eosinophils, Mast cell, T lymphocyte) in epithelial and subepithelial layer was observed and the distribution density of the cells was calculated.Results1. The percentage of eosinophils in induced sputum of EB group (10.13%(20.19)) was lower than that of asthma group ((26.00%(32.97)) (P<0.05). The percentages of eosinophils in induced sputum of EB and asthma group were significantly higher than that of control group( (0.25%(0.50) )(P<0.001).2. The percentage of eosinophils in BALF of EB group (1.63% (3.81)) was significantly lower than that of asthma group (11.75%(15.70)) (P<0.001), and the percentages of eosinophils in BALF of EB group and asthma group were significantly higher than that of control group (0.38%(1.00)) (P<0.05).3. The thickness of the basement membrane of bronchial mucosa at the gap of the lingual lobe in the asthma group (4.73μm(2.61)) was significantly greater than that in the control group (3.23μm(1.35)) (P<0.001) and the EB group {3.41μm (1.89)} (P<0.01). However, there was no difference between the EB group and asthma group (P<0.05). The thickness of the basement membrane of bronchial mucosa at the anterior section of the right and lower lobe in the asthma group (5.36μm(3.65)) was significantly greater than that in the EB group (3.53μm (3.18)) (P<0.05).4. The distribution densities of Eos in the subepithelial layer of bronchial mucosa of the lingual lobe in the asthma group (8.90(52.88) piece/mm2) were significantly higher than that in EB group (0.00(10.40) piece/mm2) and the control group (0.00(0.00) piece/mm2) (P<0.05).5.The distribution density of T lymphocytes cells in the epithelial layer of bronchial mucosa of the lingual lobe in the EB group (475.76(244.75)piece/mm2) and the asthma group (583.29(507.69) pieces/mm2) was significantly higher than that in the control group (260.71(430.31)piece/mm2) (P<0.05). There were no significant differences between the EB group and the asthma group in any site (P>0.05).6.The distribution density of mast cells in the epithelial layer of bronchial mucosa of the lingual lobe in in the EB group (168.80(314.28)piece/mm2) and the asthma group (252.78(545.68) pieces/mm2) was significantly higher than that in the control group (0.00(13.61)piece/mm2) (P<0.05). In the subepithelial layer of bronchial mucosa of the lingual lobe in in the EB group (218.44(383.65)piece/mm2) and the asthma group (197.22(208.54) pieces/mm2) was significantly higher than that in the control group (66.07(45.92)piece/mm2) (P<0.05). There were no significant differences between the EB group and the asthma group in any site (P>0.05).Conclusions1. The pathological features of the airway inflammation in EB and asthma are similar. In EB patients, it is an airway inflammatory disorder characterized by infiltration of various inflammation cells (eosinophils, T lymphocytes and mast cells).2. The intensity of airway inflammation in EB is milder, and the extent was more limited, than that in asthma.PartⅡThe Initial Proteomics Analysis Between EB and asthmaObjectiveWe found that there were a great deal of similarities in pathology and immunpathology between EB and asthma in a previous study, but there still existed many differences. Bronchoaveolar lavage fluid (BALF), which can reflect the changes of airway inflammation directly and objectively, was recognized to be an ideal sample of proteomics in the investigation of airway inflammation diseases. In this study, we used the comparative proteomics technique to explore at the molecular level differences between eosinophilic bronchitis and bronchial asthma for identifying idiotype proteins associated with the airway hyperresponsiveness and reversible obstruction.MethodsThere were five cases of EB, six cases of asthma and six cases of normal subjects respectively. Fiber bronchoscopy, bronchoalveola lavage (BAL), and the BALF were performed in the right and middle lobe. Total protein extracted from the cells in BALF was separated using two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG). After silver nitrate staining, the gel image analysis was carried out using Image Master 2D Elite 5.0 analysis software to identify the proteins differentially expressed in EB, asthma and control group. The differential expression proteins were identified by peptide mass fingerprint (PMF) using matrix-assisted laser desorption /ionization time of flight mass spectrometry (MALDI-TOF-MS), followed by MALDI-TOF/TOF-MS for a secondary PMF, and 4 protein spots were identified.ResultsAs shown in the two-dimensional electrophoresis, there were significant differences in the protein expression in BALF cells from the EB, asthma and normal control group. After comparing the peptide mass fingerprints of various protein spots with standard molecular weight and isoelectric point, two proteins, ferritin (light polypeptide) and Human Glutathione -S -Tranferase T1 (GSTT1), were identified. Ferritin was up-regulated in asthma group compared with the EB and control group. 83.3% in asthma patients and 33.3% in controls had no expression of GSTT1 , while the expression of GSTT1 was relatively higher .Conclusions1. The comparative analysis of two-dimensional electropherogram displayed that the protein expressions in the BALF cells among these three groups were distinct. Identification of the different proteins provided an important method and clue for discovering the protein markers of airway hyperresponsiveness and elucidating the pathogenesis of airway responsiveness.2. By comparative proteomics technique, we have succeeded in the identification of two differentially expressed proteins in the EB, asthma and normal group, and we found that GSTT1 might be closely related to the development of airway responsiveness. PartⅢEstablishment of Eosinophilic Bronchitis Mouse Model and Comparison in Airway Inflammation between Eosinophilic Bronchitis Model and Asthma ModelSegment1 Proper Method for Measuring Airway Responsiveness in Allergic MiceObjectiveTo investigate the applicability of invasive measurement and non-invasive measurement systems in analyzing hypersensitivity of the lower airway in mice.MethodsFemale Balb/c mice 6 weeks of age were obtained and divided randomly into two main groups: airway allergic group and normal control group. Each group was divided again into 2 sub-groups (4 groups in all), n=8, according to the different measurements: non-invasive airway allergic group, non-invasive normal control group, invasive airway allergic group and invasive normal control group. Mice were immunized by an intraperitoneal injection of 10μg OVA (Sigma, USA)1.3mg of aluminum hydroxide gel on days 0 , 7 and 14, followed by daily intranasal challenges on days 28 with 0.2% OVA, which was diluted in sterile normal saline (50 ul OVA solution, 2_ per mouse). The control mice received saline injection and intranasal challenge instead of the OVA solution. The mice were subjected to the following evaluations on days 29 (24 hours after intranasal challenge).The measurement of airway responsiveness was carried out by using invasive measurement system (“RC”system, Buxco,USA) and non-invasive measurement system (“penh”system, Buxco,USA). Measurements were made at concentrations of 0(saline), 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25and 50 mg/ml MCh. In the invasive measurement, AR was assessed as an increase in RL after challenge with aerosolized MCh in anesthetized, tracheotomized and ventilated mice. To measure transpulmonary pressure (Ptp), a water-filled tube was inserted into the esophagus to the level of the midthorax and coupled to a pressure transducer. Changes of lung resistance (RL) and lung dynamic compliance (Cdyn) were measured. In the non-invasive measurement, mice were placed into the chamber and allowed to move freely. Saline and MCh solutions of increasing concentrations were aerosolized and pumped into the chamber. Penh was measured after each aerosolization. After the measurement of airway responsiveness, bronchial alveolus lavage fluid (BALF) and histopathological evaluation of the nose and trachea was performed by a pathologist blinded to treatment of the groups. Epithelial erosions and inflammation were scored using a subjective semiquantitative scale of 0-5 as Underwood described before.Results1. At Mch concentrations of 6.25, 12.5 and 25mg/ml, Penh levels were significantly higher in the non-invasive airway allergic group than that in the non-invasive normal group. Penh200 and Penh 300 in the non-invasive airway allergic group were significantly lower than that in the non-invasive normal group (P<0.001). However, in the invasive measurements, significant bronchoconstriction was not observed in either the invasive airway allergic group or the invasive normal control group.2. In the non-invasive airway allergic group and invasive airway allergic group, no significant changes in the cell number or cell differentials in BALF were observed. Furthermore, the score of the epithelial erosions and inflammation was not different between these two groups.3. The nasal mucosa showed a mild infiltration of inflammatory cells in airway allergic groups and the mean number of nasal itching in airway allergic groups was significantly greater than that in normal control groups at 30mM-histamine concentration. ConclusionsOur study showed that invasive and non-invasive measurement might lead to the different results for airway responsiveness in allergic mice. We think one reason was that the airway inflammation of the allergic mouse was comparatively mild, and this lead to no apparent bronchoconstriction in lower airway. Another possibility was that in the non-invasive measurements, inhalation exposure includes nasal and gastro-intestinal uptake, and it might not necessarily represent a change in the lower respiratory tract. The increased airway responsiveness in the non-invasive approach is related with obstruction of upper airway. We suggest it is necessary to use the invasive measurement first to measure the lower airway hyperresponsiveness of the allergic mice.Segment 2 Establishment of Eosinophilic Bronchitis Mouse Model without HyperresponsivenessObjectiveTo establish an eosinophilic bronchitis mouse model without airway hyperresponsiveness.MethodsFemale Balb/c mice were obtained and randomly divided into 4 groups: airway allergic aerosolized group (aerosolized group), airway allergic intranasal challenge group 1(intranasal challenge group 1), airway allergic intranasal challenge group 2 (intranasal challenge group 2) and normal control group (control group). Mice were immunized as before, then the aerosolized group was aerosolized on day 28 with 0.2% OVA. The intranasal challenge group 1 was followed by intranasal challenge on day 28 with 0.2% OVA while the intranasal challenge group 2 was followed by intranasal challenge on days 28, 29 with 0.2% OVA. The Control group was sensitized and challenged with diluents. Twenty four hours after the latest challenge, the measurement of airway responsiveness was carried out using the invasive measurement system (“RC”system, Buxco, USA). After the measurement, a pathologist blinded to the treatment of the groups performed bronchial alveolus lavage fluid (BALF).Results1. At Mch concentrations of 25 mg/ml, RL% in the intranasal challenge group 2 was higher than that in the control group(P<0.05). No difference in airway reactivity between the aerosolized group and the control group and the same was found between the intranasal challenge group 1 and the control group.2. The percentage of BAL macrophages decreased significantly in all OVA immunized mice when compared with the control group and the percentage of BAL lymphocytes, neutrophils and eosinophils increased significantly in all OVA immunized mice when compared with the control group.3. The percentage of macrophages in BAL in the aerosolized group was higher than that in the intranasal challenge group 1 and group 2 while the percentage of eosinophils in BAL ((7.40±3.41)%) was lower than that in the intranasal challenge group 1 ((25.33±10.79)%)and group 2 ((39.73±6.02)%).ConclusionsBy changing the antigen immunizing and challenge methods, it is possible to establish an eosinophilic bronchitis mouse model without airway hyperresponsiveness. We achieved the goal successfully. Segment 3: The Airway Inflammation Characteristics of Eosinophilic bronchitis Mouse Model and the Comparison between Eosinophilic Bronchitis Model and Asthma ModelObjectiveTo observe and compare the airway inflammation characteristics of the eosinophilic bronchitis mouse model and the mice model of asthma.MethodsFemale Balb/c mice were obtained and divided randomly into 3 groups: eosinophilic bronchitis group (EB group), asthma group and normal control group (control group). Mice were immunized as before. EB group was followed by intranasal challenges on day 28 with 0.2% OVA and asthma group was followed by intranasal challenges on days 28, 29, 30 with 0.2% OVA. The control mice received saline injection and intranasal challenge instead of the OVA solution. Twenty four hours after the latest intranasal challenge,the measurement of airway responsiveness in every group was carried out using the invasive measurement system (“RC”system, Buxco, USA) and a pathologist blinded to the treatment of the groups performed bronchial alveolus lavage fluid (BALF) and histopathological evaluation of the trachea and lung. Twelve and 48 hours after the latest intranasal challenge,the measurement of airway responsiveness was carried out using the invasive measurement system (“RC”system, Buxco, USA) and bronchial alveolus lavage fluid (BALF) performed in EB group.Results1. Twelve, 24 and 48 hours after intranasal challenge, no difference of airway reactivity were found between the EB group and the control group. In contrast, at Mch concentrations of 12.5, 25, 50mg/ml, RL% in the asthma group was higher than that in the control group(P<0.05). 2. Twenty four hours after intranasal challenge, the percentage of BAL eosinophils and the total cell number in BALF of the asthma group ((42.15±13.35)%, (7.59±1.33)×105/ml) were higher than that in EB group ((25.33±10.79)%, (2.86±0.55)×105/ml)). Forty eight hours after intranasal challenge, the percentage of BAL eosinophils increased significantly when compared with that after 12 and 24 hours in EB group. Furthermore, the score of the epithelial erosions and inflammation in asthma group was signicantly higher than that in EB group(P<0.05)。Conclusions1. Airway inflammation in the EB model became more severe as time went by, while significant bronchoconstriction was not observed during our observation period.2. The intensity of airway inflammation in the EB model was milder than that in asthma model.更多还原