【作者】 赵清娟；

【导师】 安淑华；

【作者基本信息】 河北医科大学 ， 儿科学， 2010， 硕士

【摘要】 背景:支气管哮喘(Bronchial Asthma)简称哮喘,是儿童期最常见的慢性呼吸道疾病,是由多种细胞如肥大细胞、嗜酸性粒细胞、T淋巴细胞等参与的慢性气道炎症,临床表现为反复发作性喘息,呼吸困难、胸闷或咳嗽,全球哮喘患病率呈上升趋势。持续气道炎症使气道反复损伤、修复,将导致气道结构变化即发生气道重塑,主要表现为平滑肌细胞增生肥大,上皮下胶原沉积,杯状细胞增生,新血管生成等。其中细胞外基质(extracellular matrix, ECM)在气管上皮下的异常沉积是哮喘特征性病理改变。金属基质蛋白酶(matrix metalloproteinases, MMPs)为调节ECM代谢的主要限速酶,可降解ECM的大多数成分。金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinases, TIMP)-1可1:1与MMP-9结合,特异性抑制MMP-9的活性。MMP-9/TIMP-1失衡导致细胞外基质代谢异常,可能参与哮喘气道重塑过程。正常T细胞表达和分泌的活性调节蛋白(regulated upon activation normal T cell expressed and secreted, RANTES)属于半胱氨酸-半胱氨酸(Cys-Cys, CC)类趋化因子,又称CCL5,由多种炎性细胞分泌,它对嗜酸性粒细胞、单核细胞、树突状细胞、淋巴细胞、NK细胞等有趋化作用,尤其是对嗜酸性粒细胞强大的趋化作用,可加重哮喘气道炎症,参与气道重塑的发生。目的:通过光镜观察实验大鼠气道病理变化,计算机图像分析软件量化气道壁增厚情况,了解哮喘气道重塑情况;检测大鼠气道MMP-9、TIMP-1及肺泡灌洗液RANTES的表达水平,探讨MMP-9/TIMP-1在哮喘气道重塑中的作用,分析MMP-9/TIMP-1与RANTES的相关性。方法:按照完全随机分组法,将36只雄性Wister大鼠分为哮喘模型组(A组)、地塞米松干预组(B组)、正常对照组(C组)三组。采用卵白蛋白(Ovalbumin, OVA)和氢氧化铝的复合制剂致敏、OVA激发方法。第1、7、14天,A、B组大鼠分别给予腹腔注射10%卵白蛋白与氢氧化铝混悬液1ml/只致敏,C组则相应给予腹腔注射生理盐水1ml/只。第21天A、B组开始雾化吸入1%的卵白蛋白激发喘息发作,每次雾化吸入30分钟,隔天1次,共6周,C组雾化吸入0.9%生理盐水作为对照。B组自雾化激发后1周起雾化前半小时予尾静脉注射地塞米松0.5mg/Kg每周3次,共5周,A、C组对应给予0.9%生理盐水0.5ml尾静脉注射。三组大鼠均于末次雾化吸入后24小时进行1%戊巴比妥钠0.2ml/Kg腹腔注射麻醉开胸,结扎左肺取肺组织固定、包埋;行气管插管,用生理盐水5ml冲洗5次回收右肺肺泡灌洗液,1000转低温离心5分钟,取上清。制作左肺组织的HE染色切片,光镜观察各组切片中气道的病理学变化,多功能彩色病理图像分析仪测量切片中支气管横截面的基底膜周径(bronchial basement membrane perimeter, Pbm),气道平滑肌面积(airway smooth muscle area, WAm),气道内壁面积(inner way area , WAi),以WAm/Pbm和WAi/Pbm作为气道重塑指标;将左肺组织切片用免疫组化方法标记MMP-9、TIMP-1,并应用多功能彩色病理图像分析仪测定MMP-9、TIMP-1积分光密度(integral optical density, IOD);双抗体夹心酶联免疫吸附试验(ELISA)检测右侧肺泡灌洗液RANTES水平。采用SPSS10.0统计软件,对组间WAm/Pbm和WAi/Pbm、MMP-9和TIMP-1数据进行单因素方差分析,P<0.05为差异有统计学意义,将MMP-9/TIMP-1与RANTES做相关性分析,计算相关系数r,P<0.05有统计学意义。结果:哮喘模型组致敏的大鼠,在雾化OVA时均出现喘息发作,雾化6周后,大鼠左肺支气管HE染色的病理切片中出现平滑肌、管壁增厚的重构现象,检测切片中气道平滑肌面积/基底膜周径(WAm/Pbm)和气道内壁面积/基底膜周径( WAi/Pbm )分别为5.78±1.36μm~2/μm, 15.10±3.99μm~2/μm较正常对照组明显增大(P<0.05)。地塞米松干预组在第一周雾化OVA即出现喘息发作,在第二周应用地塞米松后喘息减轻,雾化6周后,其左肺支气管HE染色切片中气道重塑现象不明显,测量气道管壁WAm/Pbm和WAi/Pbm为3.98±1.25μm~2/μm, 9.86±2.47μm~2/μm,较哮喘模型组气道为轻(P<0.05)。哮喘模型组大鼠左肺组织中MMP-9和TIMP-1的平均积分光密度(IOD)(1.7655±1.0476, 1.958±0.0927)及右肺肺泡灌洗液RANTES表达水平(18.20±4.00?pg/ml)明显高于正常对照组、地塞米松干预组(P<0.05),地塞米松干预组MMP-9和TIMP-1的平均IOD(0.8525±0.0231, 0.876±0.0302)及肺泡灌洗液中RANTES水平(9.28±2.26pg/ml)与正常对照组无明显差别(P>0.05)。MMP-9/TIMP-1在哮喘模型比例失衡,与肺泡灌洗液RANTES水平呈负相关(r=-0.141,P<0.05)。结论:哮喘时MMP-9、TIMP-1及RANTES的表达水平均提高,可能有加重气道炎症的作用;哮喘时MMP-9/TIMP-1比例失调,与炎性细胞趋化因子RANTES水平呈负相关,与ECM的降解与沉积有关,参与气道重塑的发生;地塞米松对RANTES及MMP-9、TIMP-1表达有明显的抑制作用,减轻气道重塑。更多还原

【Abstract】 Background: Referred to as asthma, Bronchial Asthma, the most common chronic childhood respiratory disease, is a kind of chronic airway inflammation by a variety of cells such as mast cells, eosinophils, T lymphocytes, and others involving, manifested as recurrent episodes of wheezing, difficulty breathing, chest tightness or cough. The incidence of asthma shows an up trend in the global. Continuous airway inflammation in the airway induced repeatedly airway injuring and repairing, which may lead to structural changes known as airway remodeling which includes airway smooth muscle hypertrophy and hyperplasia, collagen deposition to sub-epithelial basement membrane, hyperplasia of goblet cells, and an increase in vascularity, etc. Extracellular matrix (ECM) protein deposition beneath the basement membrane is characteristic of the airway wall of an asthmatic individual. Matrix metalloproteinases (MMPs) were the main rate-limiting enzymes in ECM metabolism, which could degrade the majority components of ECM. Tissue inhibitor of metalloproteinases (TIMP) -1, which interacted 1: 1 with the domain of MMP-9, inhibited the activity of MMP-9. The imbalance of MMP-9/TIMP-1 leading to abnormal metabolism of extracellular matrix, may be involved in the process of airway remodeling in asthma. Regulated upon activation normal T cell expressed and secreted (RANTES) are cysteine - cysteine type chemokine, also known as CCL5, is a secretion of a variety of inflammatory cells, such as eosinophils, monocytes, dendritic cells, lymphocytes, NK cells and so on. RANTES, which has chemotactic effects, especially for a powerful eosinophil chemotactic role, could aggravate airway inflammation of asthma, and may also be involved in airway remodeling from occurring. Objectives: To learn the situation of airway remodeling by observing dthe rats airway pathological changes with light microscopy, and quantifiing the airway wall thickening of rats bronchi with multi-function color pathological image analysis system.To observe the expression of MMP-9, TIMP-1 in the lung tissue and RANTES in bronchoalveolar lavage fluid (BALF) of each group rats and study the correlation between MMP-9/TIMP-1 and RANTES.Methods: Thirty-six young male Wistar rats were randomly divided into three groups: asthmtic model group (Group A), Dexamethason (DEX) intervention group (Group B) and normal control group (Group C). At 1, 7, 14 days, 10% of Ovalbumin (OVA) and aluminum hydroxide [Al2(OH)3] compound preparation (1 ml) was applied to sensitize the rats in Group A and B by injection Intraperitoneally each. Group C were given intraperitoneal injection of normal saline at the same dosage as normal control. On the 21th day, the rats in Group A and B inhaled 1% of OVA atomization 30 min each time to excite wheezing, every other day for 6 weeks, Group C inhalation with normal saline instead. One week after the excitation, Group B began to be intervened via tail vein injection with DEX (0.5mg/Kg) half an hour before inhalation three times a week, Group A and C tail vein injection with normal saline. At the last meeting 24 hours after inhalation the rats of three groups were taken thoracotomy under anesthesia with an intraperitoneal injection of 1% pentobarbital sodium (0.2ml/Kg). The left lung main bronchus of the rats was ligated and cut to be fixed and embedded. The BALF of right lungs were collected.Pathological changes in left lung airway of each group were observed in the left lung organization slices. Bronchial basement membrane perimeter (Pbm),airway smooth muscle area (WAm), inner way area (WAi) were measured by multi-function color pathological image analysis system, and WAm/Pbm and WAi/Pbm taken as indicators of airway remodeling. Immunohistochemistry assays was used to determine the status of MMP-9 and TIMP-1 expression in the left lung organization slices, and mean integral ?optical density (IOD) of MMP-9 and TIMP-1 were also measured by multi-function color pathological image analysis system. The contents of RANTES in BALF of right lungs were determined by enzyme linked immunosorbent assay (ELISA).All data were processed by SPSS 10.0 statistical software. Data comparisons between groups were tested by One-Way ANOVA analysis, and the correlation coefficient of correlation analysis between MMP-9/TIMP-1 and RANTES was calculated, P <0.05 as statistically significant difference.Results: There is wheezing onset of the rats in asthmatic model group during OVA inhalation. Airway remodeling such as airway wall and smooth muscle thickening could observed in the left lung bronchial biopsy. WAm/Pbm and WAi/Pbm of the rats left airway in asthmatic model group slices are 5.78±1.36μm~2/μm, 15.10±3.99μm~2/μm, higher than that in normal control group (P<0.05). The rats in DEX intervention group wheeze after OVA inhalation in the first week, and there is a relief after DEX intervention in the following weeks. Airway remodeling in DEX intervention group was not seen. WAm/Pbm and WAi/Pbm of the rats left airway in DEX intervention group slices are 3.98±1.25μm~2/μm, 9.86±2.47μm~2/μm, lower than that in asrhmatic model group (P<0.05).The MMP-9 and TIMP-1 mean IOD values (1.7655±1.0476, 1.958±0.0927)of rats left lung and the RANTES expression (18.20±4.00 pg/ml ) of rats right BALF in asthmatic model group are significantly higher than those in normal control group and DEX intervention group (P<0.05). The MMP-9 and TIMP-1 mean IOD values (0.8525±0.0231, 0.876±0.0302) of rats left lung and the RANTES expression (9.28±2.26pg/ml) of rats right BALF in DEX intervention group are no differences with those in normal control group. The imbalance of MMP-9/TIMP-1 in asthmatic model group was negatively correlated with RANTES of BALF (r=-0.141, P<0.05).Conclusions: The expression of MMP-9, TIMP-1 and RANTES all increase conspicuously in asthma, which could promote the progress of airway inflammation. The imbalance of MMP-9/TIMP-1 in asthmatic model group was negatively correlated with RANTES, contribution to ECM degradation and deposition related to participation in the occurrence of airway remodeling. DEX could inhibite MMP-9, TIMP-1 and RANTES expression, reducing airway remodeling.更多还原