【作者】 张贺；

【导师】 李菁华； 肖纯凌；

【作者基本信息】 吉林大学 ， 病原生物学， 2013， 硕士

【摘要】 随着经济建设和工业化的进展,环境大气中颗粒污染物及多种气体污染物日渐增多。本研究通过制备大气混合污染物动物模型的方法,在病理学及分子水平上,对环境大气中所含颗粒污染物及多种气体污染物引起肺损伤的作用机制加以探索和研究,分析血红素氧合酶1（HO-1）在大气混合污染物致肺损伤时的作用机制。本实验所采用的试验动物模型,为颗粒污染物及多种气体污染物染毒大鼠，具体方法采用向大鼠进行气管内注入PM2.5混悬液进行染尘,然后,动态吸入含有SO₂、NO₂及CO的空气混合气体进行染毒,对大鼠的各种病变进行观察,从而模拟大气污染所诱导的肺损伤。观察染尘染毒后大鼠肺组织大体形态的病理学变化及分子水平变化,采用常规切片染色法观察大鼠肺组织组织病理学变化,以RT-PCR技术分析大鼠肺组织中HO-1的mRNA表达的变化,采用Western blot法分析大鼠肺组织中HO-1蛋白的表达变化,采用免疫组化方法分析大鼠肺组织中HO-1蛋白的表达部位及变化,通过测定HO-1反应系底物浓度变化,分析大鼠肺组织中HO-1蛋白活性的变化,通过上述方法,研究HO-1在大气混合污染物诱导肺损伤时的分子及蛋白水平变化,探索肺损伤时其对机体的影响和作用机制。实验结果显示,染毒组大鼠,于染毒后7d,肺组织与对照组相比出现充血、水肿、炎细胞浸润等病理变化,并持续至染毒后30d,染毒后30d大鼠肺组织出现水肿、炎细胞浸润等病理变化外,伴有部分间质纤维组织增生,染毒组大鼠中,染毒后7d开始大鼠肺组织中HO-1的mRNA表达及蛋白水平与对照组相比明显升高,并持续至染毒30d,免疫组化结果显示,染毒7d组大鼠肺泡上皮细胞及巨噬细胞呈现HO-1阳性,染毒后30d,大鼠肺泡和支气管上皮细胞持续HO-1阳性表达,同时,染毒组大鼠于染毒30d后HO-1蛋白活性比对照组明显升高。本研究提示在大气混合污染物所致大鼠肺损伤的过程中,伴随着HO-1基因及蛋白表达的增加,及HO-1蛋白活性的增强。本研究结果提示,HO-1参与大气混合污染物所致肺损伤这一病理过程,在污染物所致肺损伤7天以后,HO-1诱导性增高,结合HO-1分解血红素的生理作用和其代谢产物的影响,提示诱导HO-1增高可作为预防和保护大气混合污染物所致肺损伤的一种方法,另外,在大气混合污染物诱导肺损伤的过程中, HO-1基因及蛋白表达也可作为检测早期肺损伤的敏感性指标之一。更多还原

【Abstract】 In recent years, air pollution was increasing seriously and could induce injury tohuman health. Air pollutants were complex components, and formed from a varietypollutants. The main pollutants in the pollutant particles were the particulate matter（PM10） which diameter was less than10um. Recent studies were found that particulatematter which diameter was less than2.5um （PM2.5） could enter the lung tissue andblood deeply. PM2.5was more harmful to the human body. On the other hand, with thedevelopment of the rapid industry and people’s living standard, energy consumptionand the emission of CO、NO₂、SO₂were increased.In the heme degradation process, HO-1acts as a rate limiting enzyme andproduces bilirubin, ferritin and carbon monoxide （CO）, and the protective system ismediated by the physiologic activities of these products. Since heme oxygenase-1（HO-1） is known to protect lung tissue against various kinds of oxidative stress,increased expression of HO-1in thought to reflect oxidative stress. Expression ofHO-1is significantly induced by oxidative stress such as ultraviolet rays, heat shock,endotoxin and cytokine. Increased HO-1expression has also been reported in humaninterstitial pneumonia and cystic fibrosis.Objective:In order to determine whether HO-1were involved in lung injury caused byexposure to mixed pollutants or not. We investigated the heme oxygenase-1geneexpression in lung injury caused by exposure to simulated atmospheric pollution. Weused a rat model in which animals were exposure to simulated atmospheric pollution.We investigated the morphological changes and histological changes in the lungsfollowing exposure to simulated atmospheric pollution. Then, we analyzed theexpression of HO-1mRNA and protein in the lungs of rats, and the activity of HO-1. Methods:30Wistar rats were randomly divided into three experimental groups （3d，7d,1Mgroup） and three control groups （control3d，control7d, control1M group）.5rats eachgroup respectively. Rats were anesthetized by ether. Either1ml saline or1ml PM2.5suspension （10mg/1ml saline） was administered to the animal intratracheally. The dayafter administration, experimental animals were inhaled with air mixture of whichconcentrations of SO₂、NO₂、CO were15,12,400mg/m3for4hours everyday. Thecontrol groups were fed in normal air. Rats were killed in4d,8d and31d aftercontamination. Rats were operated and right middle lung lob were taken out. TheRNA and proteins were extracted from the lung, and the expression of HO-1mRNAand protein were detected by RT-PCR and Western blotting. The left lung was inflatedand fixed by intratracheal instillation of4%paraformaldehyde and theimmunostaining for HO-1was performed. The liver tissue of control animals wereprepared by homogenization for determine the activity of HO-1.Result:1. Histopathological findingsIn the experimental animals, after7d and30d after exposure, congestion andedema of lung tissue were observed compared with control groups. Infiltration ofinflammatory cells （mainly neutrophils and alveolar macrophages） was party observedin these experimental animals （7d and30d after exposure）. After30d exposure bymixed pollutants, proliferation of fibrous tissue was detected in the lung slide ofexperimental animals compared with control groups（p＜0.05）.2. Gene expression of HO-1mRNAIn control animals, the expression of HO-1mRNA was very low duringexperiment period. Significent increased expression of HO-1mRNA was detected inthe mixed pollutants-exposed groups after7d and30d compared with control groups（p＜0.05）.3. Expression of HO-1proteinIn control animals, the expression of HO-1protein was very low during experiment. Significent increased expression of HO-1protein was detected in themixed pollutants-exposed groups at7d and30d following exposure compared withcontrol groups（p＜0.05）.1. Immunohistochemical staining for HO-1geneIn the control groups, there are less HO-1-positive cells in the lung slides. In theexperimental groups, at7d and30d after administration, the HO-1-positive cells werefound in some alveolar and bronchial epithelial cells compared with control groups（p＜0.05）.2. Activity of HO-1proteinThe activity of HO-1protein in the mixed pollutants-exposed groups at7d and30d were higher than control groups. There were significant different betweenexperimental groups （30d） and control groups（p＜0.05）.Conclusion:1. In the normal lung tissues of rats, the expression of HO-1mRNA and proteinwere very low.2. In the experimental groups, increased expression of HO-1mRNA and proteinwas detected in the mixed pollutants-exposed groups at7d and30d after exposurecompared with control groups.3. Significant histopathological changes and higher expression of HO-1mRNAand protein were detected in the mixed pollutants-exposed groups at30d afterexposure compared with control groups. The activity of HO-1protein wassignificantly increased at this time-point.更多还原