【作者】 黄先杰；

【导师】 栾斌；

【作者基本信息】 郑州大学 ， 儿科学， 2010， 硕士

【摘要】 支气管哮喘是由多种细胞,包括炎性细胞和气道结构细胞以及细胞组分共同参与的气道慢性炎症性疾病,以气道炎症、气道重塑及气道高反应性为特点。反复的气道炎症在不断的损伤与修复过程中,逐渐引起了气道结构的改变,包括上皮细胞脱落、上皮下纤维化、气道平滑肌增厚、平滑肌层与上皮层距离缩短、黏液腺化生和杯状细胞化生、血管增生及重塑、气道壁水肿等,被称为气道重塑。气道重塑与哮喘患者的气道高反应性及不完全可逆性气流受限都有着密切的关系,其具体发生机制尚未完全阐明。基质细胞衍生因子-1(stromal cell derived factor-1,SDF-1)及CXC趋化因子受体4(CXC chemokine receptor 4, CXCR4)属于趋化因子家族,两者相互作用通过趋化炎症细胞、促进血管生成和血管重塑、调节其他细胞因子的表达等影响支气管哮喘的病理生理过程及气道重塑的发生。本实验通过建立小鼠哮喘模型,研究SDF-1及CXCR4在小鼠肺内的表达以及二者在哮喘气道重塑发生中的作用,并应用布地奈德进行干预,以期为哮喘的早期干预及治疗提供理论依据。目的本实验通过建立小鼠哮喘模型,探讨哮喘小鼠发病过程中基质细胞衍生因子(SDF-1)及其受体CXCR4在肺内表达的变化,及布地奈德的干预对其表达的影响。材料和方法1动物分组和模型制作方法雄性清洁级6-8周龄BALB/c小鼠30只,随机分为对照组、哮喘组、干预组,每组10只。哮喘组小鼠于第1天、第8天和第15天腹腔注射卵清蛋白(ovalbumin,OVA)／氢氧化铝混合液0.2ml(内含OVA 20μg,氢氧化铝1mg)致敏,从第22天开始用2% OVA生理盐水雾化吸入激发,隔天一次,每次30分钟,共激发7次；干预组以相同方法致敏和激发,每次OVA雾化激发30分钟前用1mg布地奈德混悬液雾化治疗；对照组腹腔注射及雾化吸入均用生理盐水替代,方法同哮喘组。2肺组织标本处理各组小鼠于末次雾化结束后24小时内用戊巴比妥钠45mg/kg腹腔注射进行麻醉,迅速开胸取左肺,置于液氮冻存,留作RT-PCR用,然后经右心室插管至肺动脉,用生理盐水快速冲洗后取右肺中叶,置于4%甲醛溶液固定24小时,酒精梯度脱水,石蜡包埋,做厚度5μm切片,分别做HE染色和免疫组织化学染色。3免疫组化及HE染色结果分析结果分析采用Image Pro Plus 6.0图像分析软件,对HE染色图片测定相同级别的完整的支气管横断面的支气管壁面积(μm2)、气道上皮黏膜层面积(μm2)、气道平滑肌面积(μm2),并将其与基底膜周径(μm)的比值代表气道壁厚度(μm2/μm)、上皮黏膜层厚度(μm2/μm)、平滑肌层厚度(μm2/μm)。对于免疫组化图片测定阳性区平均光密度值,每张切片选择至少5个高倍镜视野,取其平均值作为该切片的代表值。4 RT-PCR将冻存左肺用Trizol法提取总RNA,并逆转录为cDNA,设计引物扩增目的片段CXCR4与β-actin。扩增后进行琼脂糖凝胶电泳,用Band Scan 5.0凝胶分析软件分别测定CXCR4与(3-actin的灰度值,并以CXCR4与β-actin的比值作为该小鼠的代表值。5统计学方法应用SPSS 13.0进行数据分析。计量资料用均数±标准差(x±s)表示。各组样本均数比较采用单因素方差分析,两两比较采用LSD-t检验,各因素之间的相关性采用pearson相关分析,以a=0.05为检验水准。结果1病理学改变哮喘组小鼠气道壁增厚,管腔变形狭窄,气道上皮细胞脱落,杯状细胞化生,黏液分泌增多,平滑肌层明显增厚,黏膜下及管壁周围炎性细胞浸润明显。干预组上皮细胞偶有脱落,黏膜相对完整,气道壁增厚及炎症细胞浸润均明显轻于哮喘组。对照组肺组织几乎无上述病理改变。2免疫组化结果SDF-1在哮喘组的表达(0.426±0.052)显著高于对照组(0.268±0.037),经布地奈德干预后小鼠肺内SDF-1的表达水平(0.361±0.065)明显低于哮喘组小鼠,P<0.05。3 RT-PCR结果CXCR4哮喘组的表达0.829±0.027)显著高于对照组(0.607±0.124),经布地奈德干预后小鼠肺内CXCR4的表达水平(0.723±0.094)明显低于哮喘组小鼠,P<0.05。4相关性分析SDF-1表达量与气道壁厚度呈正相关(r=0.744,P<0.01)；CXCR4 mRNA表达量与气道壁厚度呈正相关(r=0.553,P<0.01)。SDF-1及其受体CXCR4参与了哮喘小鼠气道重塑过程,布地奈德干预改善哮喘小鼠的气道重塑与降低SDF-1及CXCR4的表达有关。更多还原

【Abstract】 Bronchial asthma is a chronic airway disease that involved many cells, include inflammatory cells and airway structural cells and celelular component, characterized by airway inflammation, airway remodeling and airway hyperresponsiveness.Airway was injured by the repeated airway inflammation and repaired once and again, then the airway remodeling occurred gradually. Main changes of the airway remodeling include epithelial detachment, increased airway smooth muscle (ASM) mass, decreased distance between epithelium and ASM cells, subepithelial fibrosis, mucus gland hyperplasia, goblet cell hyperplasia, proliferation and remodeling of blood vessels and airway edema. Airway remodeling is closely related to the airway hyperresponsiveness and irreversible airway obstruction, but the specific mechanisms how airway remodeling developed was not well understood. Stromal cell derived factor-1(SDF-1) and CXC chemokine4(CXCR4), which belong to CXC chemokine family, can attract inflammatory cells, promote angiogenesis and regulator other cytokine, maybe play a role in the process of airway remodeling. In this experiment we set up the mice asthma model,study the expression of SDF-1 and CXCR4 in the airway and the effect of budesonide on their expression, aim at offering effective channel and wide thinking of the treatment and earlier intervention of asthma.ObjectiveThrough building the asthma airway remodeling model in mices, to study the expression of stromal cell derived factor-1(SDF-1)and CXC chemokine receptor 4(CXCR4) in the airway and the effect of budesonide on their expression in mices with asthma. Methods1 animal group and asthma modelThirty BALB/c male mices, aged 6-8 weeks, were randomly divided into three groups:placebo control,untreated asthma, and budesonide-treated asthma,10 mices every group. The untreated asthma group were induced by intraperitoneal injection of 10% ovalbumin (OVA)suspension(containing 0.2ml normal saline, lmg aluminum hydroxide,20μg OVA)on days 1,8 and 15,and then from days 22 to 34, chanllenged by inhalation of 2% OVA aerosol every other day. The budesonide-treated asthma group received same treatment, but was additionally given an inhalation of budesonide(1 mg)before OVA challenge.The placebo control group receive the normal saline instead both on the intraperitioneal injection and aerosol inhalation stage.2 Collecting the sampleAll the mice were anesthetized by pentobarbital sodium (45mg per kg weight), then opened the chest, intubated into the pulmonary artery through the right ventricle, using normal sodium to lavage quickly, taken the middle lobe of right lung into 4% methanal to fix them, then imbed in paraffin within 24 hours. Then made 5μm thickness slice, and did HE staining and immunohistochemistry staining.3 Analysis of the HE staining and immunohistochemistry stainingUsing computer image analysis system Image Pro Plus 6.0,chosing completed cross section of brochhi, each section was quantified under a×400 objective microscope to measure the perimeter of basement membrane (μm) and area of the airway wall (μm2), area of the tracheal epithelium(μm2), area of the airway smooth muscle(μm2), then use the ratio of area to perimeter to represent the relative thickness.On immunohistochemistry staining slice, using the same analysis system, chosing five representative filed of view randomly on each slice under high power lens(×400), selecting positive area and measuring the optical density value, then calculate the mean optical density value as the last result. 4 RT-PCRUsing the trizol method to get the total RNA from the frozen left lung, then reversed transcript into cDNA.Add a specific primer and do the polymerase chain reaction to amplify the objective fragment CXCR4 andβ-actin. The agarose gel electrophoresis is conducted to display the DNA. The gel analysis system Band Scan 5.0 can be used to detect the gray scal of CXCR4 andβ-actin. The ratio of CXCR4 to P-actin represent the expression quantity of the CXCR4 in the lung.5 Statistics analysisUsing SPSS13.0 statistical package to analyze, measurement data were demonstrated by mean±standard deviation, using one-way ANOVA to compare the mean of each group, linear correlation analysis was used to assess the relation of variables, a=0.05 is the significance level.Results1 Pathologic changesIn the lung sample of mice in the untreated asthma group, we can see the airway wall thickening, lumina narrowing, epithelial detachment, increased airway smooth muscle (ASM) mass, inflammatory cell infiltration. In the budesonide-treated asthma group, thickening of airway wall and infitration of inflammatory cells were both less than those in asthma group.But there were not such changes in control group.2 Immunohistochemistry stainingThe expression of the SDF-1 was higher in the model group than that in control group and the therapeutic group of the same period,P<0.05.3 RT-PCRThe expression of the CXCR4 was higher in the model group than that in control group and the therapeutic group of the same period,P<0.05.4 linear correlation analysis The expression of SDF-1 and CXCR4 was positive correlation with the thickness of airway wall (r=0.744, r=0.553,respectively, P<0.05).ConclusionsSDF-1 and CXCR4 are tightly correlated with airway remodeling,and glucocorticoids can reduce the expression of SDF-1 and CXCR4 in the asthma airway remodeling process.更多还原