【作者】 王震；

【导师】 梅晓冬；

【作者基本信息】 安徽医科大学 ， 内科学， 2011， 硕士

【摘要】 研究背景:慢性支气管炎(chronic bronchitis)是一种全球性患病率和死亡率很高而病因及发病机制尚不完全清楚的疾病,严重危害着中老年患者的健康,目前认为是一种可防可治的疾病。危险因素包括吸烟、粉尘、刺激性气体、感染、免疫、年龄、气候等,其中吸烟是引起慢性支气管炎发生、发展的最主要等危险因素。烟熏法复制慢性支气管炎动物模型的研究已有成功应用。高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)是一种普遍存在的高度保守的核蛋白,参与DNA的转录、复制、修复以及炎症反应等。正常情况下,人类和大鼠气道内即有一定水平HMGB1表达,对呼吸系统正常功能的维持具有重要作用。在肺组织中, HMGB1具有促炎细胞因子的作用,是介导急性肺部炎症反应重要的生物学介质。HMGB1可作为一种晚期重要的炎症因子参与对炎症的调控作用,在肺损伤、肺癌、肺纤维化、COPD等肺部疾病中已有大量研究,但HMGB1在大鼠慢性支气管炎模型中的作用尚无报道。因此本研究用烟熏法模拟人类慢性支气管炎的发病过程拟检测HMGB1在大鼠模型中的表达,初步探讨HMGB1在慢性支气管炎中的作用。目的:通过烟熏法建立大鼠慢性支气管炎模型,检测大鼠模型肺组织及血清中HMGB1的表达情况,比较HMGB1在模型组与对照组两组之间的差别,初步探讨HMGB1在香烟诱导的慢性支气管炎大鼠模型中的作用。方法:将雄性SD大鼠随机分为正常对照组、吸烟模型组,每组14只。模型组大鼠置于自制密闭烟熏箱内进行被动吸烟,每天烟熏2次,每次14支,烟熏30min ,烟雾浓度约5%(v / v),两次之间相隔不少于4 h ,每周被动吸烟6d ,连续75 d,对照组正常饲养,不作上述处理,建立慢性支气管炎大鼠模型。模型成功后,光镜下观察支气管肺组织病理形态学改变,分析支气管肺泡灌洗液(BALF)细胞计数和分类,用ELISA法检测大鼠血清中HMGB1的浓度,免疫组化法检测HMGB1在肺组织中的表达。结果:模型组大鼠肺组织的病理形态改变与人类慢性支气管的特点相一致。模型组和健康对照组大鼠BALF的细胞总数、单核巨噬细胞及中性粒细胞百分比分别为(5.12±0.69×108/L、69.62±4.82%、22.62±2.47%;1.79±0.19×108/L、86.00±2.45%、7.71±1.27%),两组相比差异有显著性意义( P < 0.05)。说明慢性支气管炎大鼠模型是成功的。模型组与对照组血清中HMGB1分别为(2.41±0.31 ug/L,1.60±0.16 ug/L),与对照组相比,模型组大鼠血清HMGB1的含量显著升高( P < 0.05)。对大鼠肺组织进行免疫组化染色观察HMGB1的表达,可见HMGB1主要分布于支气管及肺泡上皮细胞,与对照组比较,模型组大鼠肺组织HMGB1的表达强度明显增强,用图形分析软件对染色结果进行分析,测量并记录每个视野HMGB1的阳性染色的平均积分光密度值( IOD) ,模型组与对照的IOD)进行比较,有显著性差异( P < 0.05)。结论:1.与对照组相比,模型组大鼠血清HMGB1的含量显著升高。2. HMGB1主要分布于支气管及肺泡上皮细胞,与对照组比较,模型组大鼠肺组织HMGB1的表达强度明显增强。3.模型大鼠的肺组织及血清HMGB1的表达显著高于对照组,结果均有统计学意义,这说明HMGB1在慢性支气管炎的大鼠模型中起到一定的作用,但其调控机制尚需深入研究。更多还原

【Abstract】 Research background:chronic bronchitis is a disease that the etiological factor and pathogenesis are not understood entirely,endangering seriously the health of elderly patients,and it is a preventable,treatable disease currently. Smoking is one of the marjor risk factors for the development of chronic bronchitis besides dust,irritating,gases ,infection,immunication,age,climate.It has been applied successfully that replicated animal model of chronic bronchitis with smoking. High mobility group box-1 protein is a highly conserved prevalent nuclear protein ,it participates in DNA transcription, replication, repairment and inflammatory reactions. It is found that a certain level of HMGB1 expressed in both the human and rat airways . HMGB1 plays an important role in maintaining the normal function of the respiratory system. HMGB1 has a role of proinflammatory cytokines in lung tissue,it mediated important acute lung inflammation biological mediator. As a late inflammatory cytokines,HMGB1 playes an important regulatory role of inflammation,There have been many research reports of HMGB1 in lung injury, lung cancer, pulmonary fibrosis and other lung diseases, but few on COPD . However,there is no report on the role of HMGB1 in chronic bronchitis. This study established a rat model of human chronic bronchitis with smoking and detected the HMGB1 expression ,discussing the role of HMGB1 in chronic bronchitis.Objective:By establishing the rat model of chronic bronchitis, detecting rat model expression of HMGB1 in lung tissue and serum and comparing the expression of HMGB1 between the model group and the control group to reveal the role of HMGB1 of chronic bronchitis in rat model induced by smoking.Methods:The male SD rats were randomly divided into normal control group and smoking model group, 14 rats in each group. Model rats were placed inside the homemade sealed box for passive smoking,smoking 2 times a day with an interval longer than 4 h, 14 sigerates each time for 30 minutes,the concentration of smog is about 5% (v / v), Six days per week,continuously for 75 days. The lung tissue section stained by HE was observed under optical microscope in order to study the alternation of histopathologic.Total cell counts and differential analysis in BALF were performed.The concentrations of HMGB1 in serum were detected by ELISA,The expression of HMGB1 in the lung tissue was detected by immunohistochemical method .Results:The athological changes in smoking model group were consistent with that of chronic bronchitis.The total cell counting, mononuclear macrophage and neutrophil percentage In BALF were 5.12±0.69×108/L、69.62±4.82%、22.62±2.47%;1.79±0.19×108/L、86.00±2.45%、7.71±1.27% in two groups respectively with significant differences between the smoking model group and normal control group ( P < 0.05). The concentrations of HMGB1 in serum were 2.41±0.31 ug/L,1.60±0.16 ug/L in model group and normal control group respectively indicating that HMGB1 in the model group significantly increased compared with the control group (P <0.05). HMGB1 was found in the bronchial and alveolar epithelial cells , it increased significantly tban that the expression intensity of HMGB1 in model group lung tissue,using graphical analysis software to analysis of staining results, measuring and recording the positive staining of HMGB1 per field, the average integrated optical density (IOD) of the model group and the control,the expressions of HMGB1 in the lungs was obviously elevated in the model group(P <0.05).Conclusions:1. The concentrations of HMGB1 in serum in the model group significantly increased as compared with the control group.2. HMGB1 was found in the bronchial and alveolar epithelial cells, compared with the control group, the expressions of HMGB1 in the lungs was obviously elevated in the model group.3. The expressions of HMGB1 in the lungs and the concentrations of HMGB1 in serum in the model group significantly increased as compared with the control group,the results were statistically significant,HMGB1 may play a role in the pathogenesis of chronic bronchitis,but the exact mechanism needs further investigation.更多还原