【作者】 周园；

【导师】 孙延波； 肖纯凌；

【作者基本信息】 吉林大学 ， 病原生物学， 2008， 硕士

【摘要】 目的大气污染已成为目前影响人类健康的主要环境问题之一。已有的文献报道仅限于某种或某类大气污染物对肺损伤机制的研究,而大气污染对肺的损伤是由多种混合污染物所致。本实验模拟大气污染现状,旨在探讨大气颗粒污染物(particulate matter,PM)和多种气体混合物对呼吸道损害的共同作用,揭示肺炎性损伤的可能机制。方法本研究通过建立大气混合污染物(PM10、SO2、NO2、CO)导致大鼠肺损伤的动物模型,应用ELISA方法测定支气管肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)中促炎和抗炎细胞因子含量的变化及观察超微结构的改变。结果在急性炎症反应的早期,染毒组大鼠BALF中细胞因子含量高于对照组。染毒1d时IL-8表达显著高于对照组(P<0.05);染毒15d、30d组细胞因子明显低于1d组,尤为显著的是IL-1、IL-6、IFN-γ、TNF-α(P<0.05)。各细胞因子间相关分析结果显示促炎因子(IL-1、IL-8)与抗炎因子(IL-13、IL-10)均呈中等程度正相关,提示IL-1是促炎因子中代表性较好的指标;抗炎因子IL-13与促炎因子IL-1和抗炎因子IL-4均呈中等程度正相关,提示IL-13是抗炎因子中代表性较好的指标。大鼠染尘染毒后,气管纤毛摆动功能减弱、丧失,使分泌物及异物清除障碍,造成炎性损伤,导致纤毛及胞体不断损伤脱失,通气不畅,并引起肺组织的炎症反应和呼吸损失,伴有早期硬化改变;肺泡上皮渐进性损伤,形态学变化与染尘染毒时间相一致,最终发展为肺实变,残存肺组织为代偿性气肿。结论在大气混合污染物所致急性炎症反应的早期,染毒组大鼠支气管肺泡灌洗液中细胞因子含量高于对照组;不同染毒时间,支气管肺泡灌洗液中细胞因子的含量1d组表达最明显,15d、30d组细胞因子的含量呈逐渐下降趋势,炎症反应趋于平稳;支气管肺泡灌洗液中促炎细胞因子IL-1、抗炎细胞因子IL-13可作为大气混合污染物所致急性炎症反应的较好标志。更多还原

【Abstract】 Objective:The global air pollution presently caused the attention from many countries across the world. The mixed air pollutants (including PM10, SO2, NO2, CO) damage the respiratory system through breath. The documents on the lung injury caused by air pollution mainly concentrated on suspended particles, waste gas and poisonous metals, occasionally, some reports dealing with the lung injury from animal experiments were found. But we have never seen the reports involving the respiratory damage caused by the interaction of ambient particle and artificial mixed air pollutants. The reports about the effect on cytokine level in animal lung and its bronchia never appeared before. The present study tries to set up an animal model to evaluate the effect on rate lung using artificial mixed air pollutants, to measure the changes of cytokine level in (Bronchoalveolar lavage fluid) BALF and lung’s microstructure, to explore the dynamic relation and significance between cytokine level and lung injury. The results of the study will provide experimental evidence for potential mechanism of lung injury caused by air pollution.Methods1. Collection of PM10PM10 was collected in winter at Shenyang Medical College. Apparatus used to collect PM10 were maid in Japan. Filtrated films were 55mm. Air flux was 20~30L/min. We collected PM10 for 2 months, 24h/d. Filtrated films were taken in -20oC environment after desiccation and meager.2. Preparation of liquid with PM10Filtrated films were washed by ultrasonic using normal saline for 20min, absolved the water and compute amount of PM10 quantified the concentration to 22.5mg/ml.3. Preparation of air mixture (SO2, NO2, CO)The air mixed pollutants were provided by Dalian Special Gas Company including higher concentration, middle concentration and lower concentration. Each concentration was 12 times higher than that of experiment. When these air mixtures were used in this experiment, they would be diluted 12 times.4. Establishment of animal model48 Wistar rats with 200~240g weight were randomly divided into 3 experimental groups (1d dose group, 15d dose group, 30d dose group) and 3 control groups (1d control group, 15d control group, 30d control group). Each group had 8 rats. Rats anaesthetized by aether through trachea. Experimental groups were injected liquid with PM10 with 1ml. Each does group’ quantity of PM10 were 22.5mg. Control groups were injected normal saline 1ml. Experimental groups inhaled air mixture of concentration, which were diluted 12 times. The concentrations of SO2, NO2 and CO were 22.5, 18, 600mg/m3 in experimental groups. 1d, 15d and 30dgroups rats separately inhaled air mixture for 1d, 15, and 30d. Control groups inhaled normal air.5. Preparing samplesOn the 1st, 15th and 30th day after inhalation of air mixed gas, we lavaged Bronchoalveolous of anaesthetized rats by aether with 37℃physiological saline. Each rat was washed 3 times, each time with 3ml. We got 9ml BALF for each rat. BALF was placed in–80℃fridge for later use.6. Measurements and methodsELISA was used to measure cytokines (IL-1、IL-4、IL-6、IL-8、IL-10、IL-13、IFN-γ、TNF-α). ELISA kit was bought from American RapidBio. American PLUS384 auto Spectrophotometer was used to measure OD. SPSS 10.0 was used to analyze the data and the pulmonary microstructure was scraned with transmission electron microscope and scaning electron microscope.Results1. Change of pro- inflammatory cytokine in BALF In different time group, the amount of cytokine was higher in experimental group than that in control group. The level of IL-8 was higher in the first day group than that in control group (P<0.05). The level of cytokines was lower in the 15th day and 30th day groups than that in the 1th day group, especially for IL-1, IL-6, IFN-γand TNF-α(P <0.05).2. Change of anti- inflammatory cytokine in BALFIn different time group, the amount of cytokine was higher in experimental group than that in control group. The levels of IL-4, IL-10 and IL-13 were higher in the 1th day group than that in the 15th day and 30th day groups (P<0.05).3. Correlation of cytokines in BALFPro-inflammatory cytokines (IL-1, IL-8) were correlated with anti-inflammatory cytokines (IL-13, IL-10) moderately, which indicated IL-1 was a better measurement in pro- inflammatory cytokines. Anti- inflammatory cytokine IL-13 correlated with pro- inflammatory cytokine IL-1 and anti- inflammatory cytokine IL-4, which indicated IL-13 was a better measurement in anti- inflammatory cytokines.4. Change of pulmonary ultrastructureWeakness, loss of bronchial fiber waver appeared after the rats were given the mixed air pollutants, which made dysfunction of cleaning wastes and got inflammation. As the experimental time going, lung became harder than before.DiscussionThrough building up the animal model of pulmonary injury, we used ELISA to test cytokines in BALF to explore the relationship between cytokines and pulmonary injury and found the changes of cytokines associated with lung injury.In the early stage of acute pulmonary inflammation, levels of cytokines is higher in experimental groups than in control groups, which shows mixed air pollutants harm the alveolar epidermis.The levels of IL-4, IL-10 and IL-13 are higher in the 1th day group than in 15th and 30th day groups, which shows the amounts of anti-inflammatory cytokines declines with pro-inflammatory cytokines’declination.ConclusionsThrough our research we got the following evidences.1. In the early stage of acute inflammation caused by mixed air pollutants, amount of cytokine in BALF higher in experimental group than in control group.2. The level of cytokine is highest in the 1th day group, and then declines in the 15th day and in the 30th day group.3. The study shows that cytokines associates with acute pulmonary injury closely. Pro-and anti- inflammatory cytokines (IL-1 and IL-13) are the better measurements for indication of acute pulmonary injury.更多还原