【作者】 郑鸿翱；

【导师】 何韶衡；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 研究背景慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)由于患病人数多,死亡率高,社会经济负担重,目前居全球死亡原因的第4位,世界银行/世界卫生组织公布,至2020年COPD将位居世界疾病经济负担的第5位。COPD是涉及小气道和肺实质的慢性炎症,现在还没有特效药物能遏制COPD病情的进行性发展,目前临床上常把治疗哮喘的药物用于治疗COPD气道和肺实质的慢性炎症,疗效不理想。尽管COPD已成为全球重要的公共卫生问题,但是到目前为止,COPD的研究进展仍然十分缓慢,其主要原因之一是COPD发病机制复杂且缺乏可靠的动物模型,可靠的动物模型不仅有利于了解COPD发病机制,而且有利于发现新疗法。因此,需要建立可靠的动物模型以了解COPD相关的发病机制,从而对其进行干预和治疗。吸烟是气道高反应性和COPD的主要危险因素,本研究采用长期香烟烟熏方法建立COPD动物模型,并探讨细胞因子、炎性细胞等在COPD发病机制中的作用,为COPD的临床治疗提供理论基础。研究目的(1)建立稳定的烟熏COPD大鼠模型,从而为该病的进一步研究提供实验基础;(2)动态地观察整个模型建立过程中大鼠病情的变化,并探讨指标的改变在COPD形成过程中的动态变化;(3)探讨细胞因子、炎性细胞等在COPD发病机制中的作用,为COPD气道重塑的临床治疗提供理论基础。研究方法(1)将132只动物分成正常对照大鼠(正常组)和吸香烟大鼠(CS组),应用烟熏的方法复制COPD动物模型;(2)采用强迫震荡技术检查大鼠肺功能;(3)处死动物时,收集动物血液及左侧支气管肺泡灌洗液(BALF),采用酶联免疫吸附法检测血清和BALF中白介素-2、4、6、10、13、18, IFN-γ,单核细胞趋化蛋白(MCP-1)、内皮素(ET)-1的含量;(4)将收集到的全血和BALF进行细胞学分类计数和检查;(5)采用ELISA法测定大鼠气管培养液中粘蛋白含量;(6)将右肺用福尔马林固定后制备成病理标本,分别采用HE、AB-PAS、V.G染色观察支气管和肺组织病理学变化;(7)定量测定气管上皮粘液储备指数、气管壁厚度与气管外径比值、支气管胶原纤维面积与基底膜周长比值、平均肺泡内衬间隔、平均肺泡面积和全肺炎性细胞数;(8)免疫组化法观察肺内CD3、CD4、CD8和CD68的表达情况。研究结果(1)烟熏大鼠体重增长较正常大鼠明显延缓,烟熏8周起与正常组相比差异显著(P<0.05);(2)肺功能测定结果显示CS 36wk组的Raw和Ers明显增高,与正常组相比差异显著(P<0.05)。(3) CS组大鼠气管、支气管及肺组织有慢性支气管炎、阻塞性肺气肿的特征性病理改变,总体表现为纤毛上皮部分剥脱,支气管上皮和成纤维细胞增生变厚,粘液腺及杯状细胞增生,管腔内粘液聚集,气道壁和小血管周围肺泡区以巨噬细胞和淋巴细胞为主的炎细胞浸润。烟熏10周后纤毛上皮异常并伴随粘液高分泌;烟熏12周后可见气道纤毛上皮部分剥脱;烟熏12-24周后可见一些肺泡融合形成见肺大泡,烟熏24-36周后上述病理改变更加明显,尤其在烟熏36周后肺泡腔明显扩大,气道管壁明显增厚和纤维化,并可见大量炎细胞浸润。(4) CS组MLI、MAA明显高于正常组,烟熏12 wk前(含12 wk)两组比较无显著性差异(P<0.05)。CS组大鼠烟熏24 wk时MLI(68.7±2.8μm)、MAA(31.07±1.28μm)分别比正常组增加24.6%和22%,36 wk时MLI(82.0±2.3μm)、MAA(36.53±1.09,×102μm2)分别比正常组增加44.8%和43.7%;(5) CS组大鼠肺组织炎症细胞显著高于正常组(均p﹤0.01),计数值增高幅度和正常组各时点比较达16-29倍,炎症细胞以淋巴细胞、巨噬细胞为主;(6) CS组大鼠肺组织气道管壁较正常组大鼠明显增厚,气管壁厚度与气管外径比值随烟熏时间的延长呈逐步上升趋势,CS 8wk、CS 10wk、CS 12wk、CS 24wk、CS 36wk与正常对照组相同时点相比有显著性差异(P<0.05 or P<0.01);(7)大鼠肺组织V.G染色显示结果显示,CS组各类大小气道V.G染色胶原较正常组明显增加,胶原纤维面积与基底膜周长比值随烟熏时间的延长呈逐步上升趋势,CS 6wk、CS 8wk、CS 10wk、CS 12wk、CS 24wk、CS 36wk与正常对照组相同时点相比有显著性差异(P<0.05 or P<0.01);(8)正常对照组大鼠气管气管上皮内粘液阳性染色区域较小,且染色程度也浅。CS组大鼠气管上皮内粘液阳性染色区域明显增多,染色程度增强,CS组气管上皮阿辛蓝-过碘酸希夫阳性染色面积与气管上皮总面积比值(即上皮粘液储备指数)随烟熏时间的延长呈逐步上升态势,CS 4wk、CS 8wk、CS 10wk、CS 12wk、CS 24wk、CS 36wk与正常对照组相同时点相比有显著性差异(P<0.05 or P<0.01)。(9)大鼠气管环培养液中粘蛋白含量与培养时间成正比,CS组大鼠粘蛋白与正常组相比明显增加,尤其以CS 24 wk、CS 36 wk组大鼠为显著,且随着被动吸烟时间的延长而增加。气管环培养1 hr,CS 24 wk(8.74±1.04,ng/mg,下同)、CS 36 wk(9.14±1.07)比正常组增加约2.5倍和4倍;气管环培养3 hr,CS 24 wk(10.17±3.74)、CS 36 wk(21.07±3.14)比正常组增加约1.5倍和7.5倍;气管环培养6 hr,CS 24 wk(22.00±6.04)、CS 36 wk(32.32±6.59)比正常组增加约1.6倍和11.3倍;气管环培养16 hr,CS 24 wk(93.99±16.20)、CS 36 wk(114.59±13.66)比正常组增加约4倍和41倍;(10)与正常组相同时点比较,CS组在12 wk时仅有尘细胞绝对数增高(P<0.05),在24 wk、36 wk时中性粒细胞、淋巴细胞、尘细胞绝对数明显增高(P<0.05或P<0.01),增高幅度分别达正常的8.3、2.3和9.9倍,中性粒细胞、淋巴细胞、尘细胞在其余相同时点两组比较无显著差异(P>0.05)。CS组与正常组相同时点比较,巨噬细胞、嗜酸性细胞和上皮细胞的变化无显著差异(P>0.05)。与正常组相同时点比较,CS组血中中性粒细胞、淋巴细胞、巨噬细胞、嗜酸性细胞和肥大细胞的绝对数在所有相同时点的变化无显著差异。(11) CS组大鼠各时点血浆中IL-10的平均值均高于正常组,烟熏2 wk、6 wk、8 wk、10 wk、36 wk两组比较有显著性差异(P<0.05或P<0.01);BALF中IL-10测量结果相反,烟熏4 wk(19.1±4.6 pg/ml)、24 wk(23.9±3.7 pg/ml)、36 wk(17.5±3.7 pg/ml)分别比正常组下降56%、38.5%和59% (P<0.05或P<0.01);(12) IL-13测量结果显示,两组比较只有烟熏36 wk有显著性差异(均P<0.01),其中CS 36wk血浆(23.3±4.4 pg/ml)、BALF(58.9±9.1 pg/ml)分别比正常组下降75.3%和54.5%;(13)血浆IL-18测量结果显示,两组比较只有烟熏36 wk有显著性差异(P<0.01),CS 36 wk(25.5±4.7 pg/ml)比正常组增加188%;除CS 6 wk外,CS组大鼠其它时点BALF中IL-18均高于正常组(P<0.01) ;(14) CS 24 wk(16.2±2.6 pg/ml)、CS 36 wk(6.4±0.99 pg/ml)BALF中IFN-γ含量比正常组增加149%和103% (P<0.01)。(15) CS 24 wk(22.8±4.47 pg/ml)、CS 36 wk(28.36±4.26 pg/ml)BALF中MCP-1含量比正常组增加59%和85.8% (P<0.01) ;(16) CS 24 wk(22.2±2.2 pg/ml)、CS 36 wk(35.4±4.75 pg/ml)大鼠血浆ET含量比正常组增加5.5和10.7倍(P<0.01),其它时点两组比较没有显著性差异(P>0.05);BALF中ET测量结果显示,两组比较没有显著性差异(P>0.05)。(17) IL-2测量结果显示,BALF中CS 10 wk、CS 12 wk、CS 24 wk、CS 36 wk比正常组升高(P<0.05或P<0.01)。(18) IL-4测量结果显示, BALF中CS 24 wk、CS 36 wk比正常组升高(P<0.01)。(19) IL-6测量结果显示,BALF中CS 12 wk、CS 24 wk、CS 36 wk比正常组升高(P<0.01)。(20)肺组织免疫组织化学结果显示,各时点CD3+、CD8+、CD68+细胞的平均数比正常组升高,与正常组相同而时点比较,CS 2 wk、CS 8 wk、CS 12 wk、CS 24 wk、CS 36 wk的CD3+细胞比正常组升高(P<0.05或P<0.01),CS 2 wk、CS 8 wk、CS 24 wk、CS 36 wk的CD8+细胞比正常组升高(P<0.05或P<0.01),CS 2 wk、CS 4 wk、CS 6 wk、CS 12 wk、CS 24 wk、CS 36 wk的CD68+细胞比正常组升高(P<0.05或P<0.01),CD4+细胞两组之间没有显著性差异(P>0.05)。结论(1)采用熏香烟36周的方法制备大鼠COPD模型,所建大鼠COPD模型符合人类COPD的特点,说明COPD复制成功,这将有助于探究COPD的发病机制和开发药物;(2)多种炎症细胞参与了COPD的发生及发展,气道腔内炎症细胞以中性粒细胞、巨噬细胞、淋巴细胞为主,肺实质以巨噬细胞(CD68+)、T淋巴细胞尤其是CD3+、CD8+细胞浸润为主;(3) IL-2、4、6、10、13、18, IFN-γ,单核细胞趋化蛋白(MCP-1)、内皮素(ET)-1在COPD气道炎症中起重要作用;(4)气道粘液高分泌、炎症细胞增多、气道壁增厚且以胶原为主的细胞外基质过度沉积是COPD气道重塑的重要病理改变,是引起阻塞性通气功能障碍的主要原因。更多还原

【Abstract】 Chronic obstructive pulmonary disease (COPD) is one of the major causes of chronic morbidity and mortality throughout the world. It is currently the fourth leading cause of death in the world and is projected to rank the fifth as a worldwide burden of disease by 2020. It is characterized by chronic inflammation throughout the airways and parenchyma. No currently available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma.Although COPD is a major public health problem in the world, but it receives relatively little attention compared with diseases with a similar impact such as coronary heart disease and cancer. One of the main reasons is not only complex pathogenesis of COPD but also lack of reliable model of COPD.Reliable model of COPD will not only increase our understanding about pathogenesis, but will also facilitate the development and introduction of new therapeutic strategies. Therefore, more research is needed to develop reliable model of COPD and to understand the cellular and molecular mechanisms of COPD to aid the development of new therapies.Cigarette smoking is a risk factor for the development of airway hyperresponsiveness and chronic obstructive pulmonary disease. In this study, we examined the possibility of developing an animal model of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke and to study the role of inflammatory cell, cytokine in the chronic airway inflammation of COPD.Study objective(1) The present study was conducted to determine whether chronic whole-body exposure of rats to sidestream cigarette smoke could produce significant, time-related increases in the incidence of COPD, and to establish an experimental model for smoke-induced COPD. (2)To investigate the character of airway inflammation in chronic obstructive pulmonary disease (COPD). (3)To study the role of cytokine in the chronic airway inflammation of COPD.Methods(1) 132 rats were randomly divided into normal control or cigarette smoke (CS) rats. The rat models of COPD were established by exposure to cigarette smoke daily for 36 weeks (2 h/day, 7 days/week). For each CS group, air control rats were studied at the same time (all n = 7, respectively). (2) At the end of 2, 4, 6, 8, 10, 12, 24 and 36 week of smoke treatment, a forced oscillation (FO) technique independently estimated the lung function. (3) Rats were sacrificed and the levels of interleukin(IL)-2, IL-4, IL-6, IL-10, IL-13, IL-18, interferon (IFN)-γ,endothelin-1 (ET-1), monocyte chemotactic protein -1 (MCP-1) in bronchoalveolar lavage fluid (BALF) or serum were measured by enzyme-linked immunosorbent assay (ELISA). (4) Total and differential cell counts in BALF and blood were done. (5) Enzyme-linked immunosorbent assays (ELISA) for mucin were performed on culture medium after tracheas were incubated. (6) Tissue was fixed by infiating with formalin from right lung. Sagittal seetions were stained with hematoxylin-eosin or AB-PAS or V.G. (7) The pathomorphological changes, including epithelial cell mucus occupying ratio (MOR),the ratio of the wall thickness to the external diameter in the bronchus,the ratio of collagen area per unit of length of basilemma in the bronchus,mean linear intercept (MLI),mean alveolar airspace (MAA) and inflammatory cell counts (ICC) in rat lung tissues were measured with image-analysis system. (8) The expression of CD3, CD4, CD8 and CD68 in pulmonary tissue was observed by immunohistochemistry technique.Results1. The development of weight in CS rat were significantly inhibited compared with normal control.2. The increase of Raw and Ers in group CS 36wk were significantly differences than those in group normal control (P<0.05).3. The CS rats shared specific pathological features in trachea,bronchi and lung tissues with that of human chronic bronchitis and emphysema. It shows that the proliferation epithelial cell and fibroblast, epithelial layer rising into the lumens goblet cell and mucus gland significantly increased, mucus accumulation in lumens. An extensive macrophages and lymphocytes in filtrate around the pulmonary blood vessels and airways was seen in the lung interstitium of CS rats compared with normal control animals. From 10 wk CS exposure, irregular cilia arrangement of bronchial epithelium appeared in the rat bronchus. The evidence for mucus hypersecretion was also observed. The detachment of bronchial epithelium and merged cilia was found in the bronchus of CS rats after 12 wk exposure. From 12 to 24 wk CS exposure, air spaces were much enlarged in an irregular manner, some alveoli were merged and bullae were formed. Following 24 wk CS exposure, the pathological evidence of bronchitis became more apparent with hypersecretion of mucus, detachment of bronchial epithelium, merged cilia, irregular arrangement of bronchial smooth muscles and fibrosis on basement membrane. At 36 wk CS exposure, air spaces were markedly enlarged, the thickness of air walls was greatly increased,and large number of accumulated inflammatory cells was observed. The clear characteristics of chronic bronchitis including hyperplasia of bronchial epithelial cells, hypersecretion of mucus and development of peribronchial fibrosis were found at 36 wk CS exposure.4. Using quantitative histomorphology techniques, it was found that Lm and AIA of CS rats increased respectively by 24.6% and 22% after 24 wk inhalation, and by 44.8% and 43.7% after 36 wk inhalation.5. It appeared that up to approximately 29 fold increase in the number of inflammatory cells being observed in the lungs of CS rats.6. Morphometric Analysis of the aiways the thickness of each category airway walls in CS group were significantly greater than in normal control group. There was a progressive trend to the ratio of the wall thickness to the external diameter in the bronchus of CS exposed and normal control rats.7. Significant increase in thickening of collagen and the ratio of collagen area per unit of length of basilemma in the bronchus were found in CS group (P<0.05 or P<0.01) than that of normal control group.8. Stained mucin area on trachea epithelium was faint in color and small in size in normal group. Stained mucin area of CS group on trachea had the apparent increased optical density and the epithelial cell mucus occupying ratio (MOR) after 4, 8, 10, 12, 24 and 36 week inhalation were higher than those of control group respectively.9. Approximately up to 2.5 or 4 fold increase in mucin release was achieved following 1 hr incubation period. Approximately up to 1.5 or 7.5 fold increase in mucin release was achieved following 3 hr incubation period. Approximately up to 1.6 or 11.3 fold increase in mucin release was achieved following 6 hr incubation period. After 16 hr incubation, mucin secretion was elevated by 4 or 41 fold in comparison with the secretion from normal control rats.10. Compared with Sham group rats, up to approximately 8.3, 2.3 and 9.9 fold increase in the number of neutrophils, lymphocytes and dust cells was observed in the BALF of CS rats following inspiring cigarette smoke for 24 or 36 wk. At 12 wk cigarette smoking, however, only the number of dust cells was elevated in the BALF. Cigarette smoking had little effect on the number of macrophages, eosinophils and epithelial cells in the BALF of CS rats. The number of neutrophils, lymphocytes, monocytes, eosinophils and basophils in rat blood was not significantly altered after up to 36 wk cigarette smoking.11. Statistic analysis showed that level of IL-10 in serum of CS rats after 2, 6, 8, 10 and 36 week inhalation were higher than those of control group respectively,while that level of IL-10 in BALF decreased respectively by 56, 38.5 and 59% after 4, 24, and 36 wk inhalation.12. The level of IL-13 in serum and BALF decreased respectively by 75.3 and 54.5% after 36 wk inhalation.13. The level of IL-18 in serum of CS 36 wk rats was increased to 188% of control values. The level of IL-18 in BALF of CS rats were higher than those of control rats after 2, 6, 8, 10 and 36 wk inhalation.14.The level of IFN-γin BALF of CS rats increased respectively by 149 and 103% after 24 and 36 wk inhalation.15.The level of MCP-1 in BALF of CS rats increased respectively by 59 and 85.8% after 24 and 36 wk inhalation.16. It appeared that up to approximately 5.5 and 10.7 fold increase in concentration of ET in serum after after 24 and 36 wk inhalation.17. The level of IL-2 in BALF of CS rats were higher than those of control rats after 10, 24 and 36 wk inhalation.18. The level of IL-4 in BALF of CS rats were higher than those of control rats after 24 and 36 wk inhalation.19. The level of IL-6 in BALF of CS rats were higher than those of control rats after 12, 24 and 36 wk inhalation.20. Statistic analysis showed that the number of CD3+ (CS 2 wk, CS 8 wk, CS 12 wk, CS 24 wk, CS 36 wk)、CD8+ (CS 2 wk, CS 8 wk, CS 24 wk, CS 36 wk)、CD68+ (CS 2 wk, CS 4 wk, CS 6 wk, CS 12 wk, CS 24 wk, CS 36 wk) were higher than those of control group respectively, while the number of CD4 in lung parenchyma was not significantly altered after up to 36 wk cigarette smoking. Conclusions(1)The COPD models were made by the rats exposed to cigarette smoke for 36 weeks. This experimental model replicates many of the features of human chronic obstructive pulmonary disease and should facilitate studies of pathogenetic mechanisms and of potential therapeutic agents.(2) The results suggest that many inflammatory cells involve in the development and progress of COPD. The infiltration of inflammatory cells involve neutrophils, monocytes and lymphocytes in lumina of airways, and CD3+、CD8+、CD68+ in lung parenchyma.(3) IL-2、4、6、10、18, IFN-γ, MCP-1 and ET-1 might play important roles in the airway inflammation of COPD.(4) Hypersecretion of mucus, increases in inflammatory cells, increases in airway wall thickness and collagen deposition diffusely within the airway wall lead to small airway remodeling and played important role in the airflow obstruction.更多还原