【作者】 常云峰；

【导师】 文继舫； 蔡继峰；

【作者基本信息】 中南大学 ， 基础医学， 2013， 博士

【摘要】 背景镉是一种蓄积性毒物,一般情况下镉在地球表面低浓度存在,但是自然界中存在的镉很难被降解,随着工业污染的加重在环境中聚集。人类的镉暴露途径主要是通过职业接触或通过消化道食入镉污染的食物或水,镉在人体内的半衰期长达16-30年,是对人类和其它动物各脏器均有损害,且具有致癌性,早在1987年,国际癌症研究协会(IARC)把镉定为ⅡA级具有致癌性的物质,又在1993年,镉被修订为ⅠA级具有致癌性的物质,即对人类具有致癌性的物质。国际劳动卫生重金属委员会和联合国环境规划署(DNFP)把镉列为第6位对人体健康有严重危害的环境中的有毒物质,其危害性仅次于黄曲霉素和砷。镉被广泛用于电镀、油漆、颜料、电池、照相材料等工业,随着工业的发展,含镉的毒性物质只会越来越多,镉对人类健康损害及环境所造成的破坏日益严重。镉被美国农业委员会列为目前农业环境污染物中最重要的一种。镉引起的环境污染也受到世界各国高度重视。除外职业性镉接触及饮用或食入镉污染的水源和动植物导致镉在体内蓄积外,吸烟是人体接触镉并引起镉在体内蓄积的主要途径。镉对人体损害的研究目前多集中于镉致肾损害的研究,镉对肺部损害的研究报道较少,我们在对镉中毒患者死亡后尸体解剖时发现死者肺组织广泛纤维化,在我们的实验中将通过前期动物实验验证镉是否可以导致实验动物肺纤维化。在此基础上进一步通过体外细胞培养实验研究镉引起肺纤维化的分子机制。肺纤维化是多种病因引起的以肺间质进行性纤维化,呼吸功能逐渐下降为主要表现的一组肺间质性疾病,对人体危害严重,从确诊肺纤维化后患者生存时间为2-5年,5年存活率仅为20%,且到目前为止无有效的防治措施,相关统计发现随着工业对环境污染的加剧肺纤维化发病率逐年增高。国内外研究者期望通过对肺纤维化机制的研究入手,找到治疗肺纤维化的新方法。随着现代分子生物学新的研究方法和技术的不断更新,近年来对肺纤维化机制的研究也不断深入,但是肺纤维化发生机制比较复杂、影响因素又多,目前仍然没有突破性进展。已有研究发现上皮细胞-间质转化(EMT)与肺纤维化成纤维细胞聚集密切相关,EMT是一个复杂的阶梯式过程,主要特征是细胞失去上皮细胞极性和上皮细胞的特异性标记物E-钙粘素(E-cadhering, E-cad),以及获得间质细胞特异性标记物α-平滑肌肌动蛋白(α-SMA)和波纹蛋白(Vimentin)等,且细胞形态由立方形变成梭形。EMT转分化方式可能与多条信号通路有关,上皮细胞生长因子受体(EGFR)主要位于上皮细胞质膜上,很多研究表明上皮生长因子受体与肺纤维化过程中成纤维细胞和肌成纤维母细胞过度活化及增殖有关。气管及支气管上皮细胞及临近的间充质细胞在刺激物作用下可产生上皮生长因子,与上皮细胞、成纤维细胞、肌成纤维细胞和平滑肌细胞膜上的EGFR结合,可导致上皮细胞间质转化。目的对镉引起肺纤维化分子机制的初步研究,为临床预防、治疗镉引起的肺纤维化及镉引起其它人体损害的治疗提供新的思路和治疗靶点,也有助其它原因引起的肺纤维化治疗。方法1.动物实验：经呼吸道吸入及腹腔注射两种途径,给予实验大鼠高、中、低剂量的镉；经68天后将未死亡大鼠统一断颈处死并行尸体解剖(提前死亡的大鼠,即时进行解剖),记录大体解剖情况,及肺组织HE染色观察镉引起肺部损伤的形态学改变；取部分组织冻存备用。2.通过对实验大鼠肺组织进行免疫组织化学染色,观察肺组织中上皮细胞特异性标记物E-cadherin及间质细胞特异性标记物α-SMA的定位及表达情况。3.体外细胞培养实验：体外培养肺泡Ⅱ型上皮细胞(A549),用10mM/LCdC12处理生长良好的体外培养的肺泡Ⅱ型上皮细胞,于24h、48h、72h观察处理后的肺泡Ⅱ型上皮细胞形态学变化。4.荧光定量PCR检测a-SMAmRNA和E-cad mRNA的表达情况：通过荧光定量PCR检测随镉处理时间的延长时a-SMA mRNA和E-cad mRNA在肺泡Ⅱ型上皮细胞中的表达情况。5. Western blot检测a-SMA、E-cad蛋白：Western blot检测随镉处理时间的延长时a-SMA、E-cad在肺泡Ⅱ型上皮细胞中表达的变化。6.Western blot检测磷酸化的EGFR(p-EGFR)表达情况,p-EGFR代表EGFR受体途径激活。结果1.对呼吸道雾化吸入及腹腔注射不同剂量的镉的实验大鼠进行大体解剖发现均可引起不同程度的肺部损伤——肺部炎症、肺淤血、肺水肿伴肺间质纤维组织增生,其中经呼吸道吸入组肺部炎症及纤维组织增生比腹腔注射组严重。2.免疫组织化学染色观察到,间质细胞特异性标记物α-SMA在镉注射组及吸入组大鼠肺泡Ⅱ型上皮细胞阳性表达；上皮细胞特异性标记物E-cad在镉注射组及吸入组大鼠肺间质部分成纤维细胞阳性表达(此部分成纤维细胞可能来源于发生了EMT的肺泡Ⅱ型上皮细胞)。3.倒置相差显微镜观察用10mM/LCdCl2处理生长良好的体外培养的肺泡Ⅱ型上皮细胞,于24h、48h、72h观察处理后的肺泡Ⅱ型上皮细胞形态学改变,发现细胞由原来的扁平多边形变为梭形,形态如同纤维细胞,而且细胞间隙变大,细胞间离散。4.荧光定量检测a-SMA mRNA和E-cad mRNA的表达情况发现：随镉处理时间的延长肺泡Ⅱ型上皮细胞间质细胞特异性标记a-SMA mRNA表达上调,并随镉处理时间的延长而表达量呈增加趋势,而上皮细胞标记E-cad mRNA在肺泡Ⅱ型上皮细胞中的表达下调。5. Western blot检测发现随镉处理时间的延长体外培养的大鼠肺泡Ⅱ型上皮细胞间质细胞标记物a-SMA的表达增加,上皮细胞标记物E-cad表达减少。6. Western blot检测发现随镉处理时间的延长体外培养的大鼠肺泡Ⅱ型上皮细胞表皮生长因子受体EGFR及其活化形式p-EGFR蛋白表达增加。结论1.动物实验及镉中毒死亡案例镜下改变结果均证实经呼吸道吸入和腹腔注射CdCl2均可导致肺纤维化,其中呼吸道吸入组肺部炎症及纤维组织增生程度较腹腔注射组严重。2.体内镉诱导的肺炎性损伤及纤维组织增生,肺组织中发现了肺泡Ⅱ型上皮细胞发生EMT,并转化为成纤维细胞。3.体外细胞培养实验证实在镉的刺激作用下部分肺泡Ⅱ型上皮细胞可发生EMT。4.肺泡Ⅱ型上皮细胞间质转化过程中间质细胞特异性标记物a-SMA mRNA表达上调,而上皮特异性标记物E-cad mRNA表达下降。该结果支持了EMT参与镉致肺纤维化假设。5.镉可诱导EGFR受体信号传导通路的活化,可能与镉诱导的EMT及肺纤维有关。更多还原

【Abstract】 BackgroundCadmium (Cd) is a kind of accumulation poison, it is normally found at a low concentration in the soil, and it is not biodegradable in the environment through the aggravation of industry pollution. Humans are exposed to Cd in the work place or through ingestion Cd-contaminated foods or water, the half life of Cd lasts for sixteen to thirty years in human body, it can accumulate in various organs and make damages and it is even carcinogenic. Cd has been classified as the sixth toxic substance harmful to human health by United Nations Environment Programme (UNEP) and the Committee of International Public Health of Heavy Metals, second only to aflatoxin and arsenic. In1987,International Agency for Research on Cancer(IARC) ranked Cd as ⅡA Carcinogens, and amended it to ⅠA in1993.Cd was extensively used in electroplate, paints, battery and photograph industry production. With the development of industry, the environmental pollution caused by toxic substance containing Cd becomes more and more severe. The American Committee of Agriculture has ranked Cd as first-line Agriculture environment pollutant; and the environment pollution brought by cadmium has attached great importance around world. Pulmonary fibrosis is a suite of pulmonary mesenchymal disease characteristic as progressive fibration of pulmonary mesenchymal and the fall-off of the respiratory function caused by varies factors, and usually harmful to human body. After final diagnosis, the survival time of it is only12-5years, five years survival rate is20%, and there is no effective prevention and treatment. Statistics shows that, with intensify of encironmental pollution, the incidence rate of pulmonary fibrosis is increased. With the development of modern research technics, scholars want to find out new method based on the mechanism study of pulmonary fibrosis. Although there has been some progress but no breakthrough has been achieved because of the complicated mechanism and numerous influencing factors. Besides the occupational expose to Cd and ingestion the polluted water or plants, smoking is another main route of non-occupational expose to Cd.In present, most of the studies about the damage caused by Cd to human body focus on the kidney injurious while there are insufficient studies reporting the lung injury. After the finding of extensive lung fibration in an autopsy dead from Cd poisoning, we tested this phenomenon through animal experiment. In our experiment, the animal exposed to Cd was found with widespread lung fibration, based on finding of this autopsy and animal experiment, we carried out a series of animal experiments and extro cell culture in order to interpret the molecule mechanism of Cd induced lung fibration. Epithelium-mesenchymal translation (EMT) has intimate relationship with fibroblast cell aggregation in pulmonary fibrosi. EMT is a complicated multi-step progress, the key feature is epithelium lost cell polarity and epithelial specificity maker E-cadhering(E-cad), got mesenchymal specificity maker α-SMA and Vimentin, and the shape of cell changed from cube to spindle-shaped, there is several signal pathway can have relationship with EMT, of the total epithelium growth factor receptors (EGFR) pathway play an important role. ObjectiveFrom the molecule mechanism study of Cd-induced lung fibration, we try to provide a new method for the prevention and therapy of the pulmonary fibrosis and lesion caused by Cd, and we also try to offer new thought in the study of lung fibration caused by other reasons. Methods1. Animal experiment:The rat is exposed to Cd through inhalation from respiratory and peritoneal injection method, we observe the gross anatomy of lung tissue, and the micro morphology change after HE dyeing.2. Followed with the immunohistochemisty dyeing to determination the localization and expression of E-cad and a-SMA in lung tissues. 3. Cell culture test in vitro, the cultered type Ⅱ penumonocyte (A549) was treated with10mM/LCdCl2, observe the cell morphology change after24h,48h and72h.4. Fluorescent quantitation testing the expression of a-SMA mRNA and E-cad mRNA in cultered type Ⅱ penumonocyte (A549) which have been treated with10mM/LCdCl2for24h、48h、72h.5. Western blot testing the change of a-SMA and E-cad expression in cultured cell be treated with10mM/LCdCl2for24h、48h、72h.6. Western blot testing the p-EGFR, which represents the activation status of EGFR signaling pathway. Results1. Lung injuries in gross autophy is found in both the rat exposed to CdCl2through respiratory inhalation or peritoneal injection, and the micro observation after HE dyeing shows lung inflammation, lung congestion, lung hydrops and pulmonary fibrosis, and in the group exposed to CdCl2through respiratory is more grievous.2. Observation after immunohistostaining in both two groups mesenchymal specificity maker a-SMA is high positive expression and epithelial specificity maker E-cadhering (E-cad) is positive expression in type Ⅱ penumonocyte, epithelial specificity maker E-cadhering (E-cad) is positive expression in part of the fibroblast in interstitial tissue, this phenomenon gives the hint that this part of fibroblast comes from type Ⅱ penumonocyte.3. Observation of the cultured type Ⅱ penumonocyte cell treated by lOmM/L CdCl2after24h,48h and72h under inverted phase contrast microscope, the shape of type Ⅱ penumonocyte is changed from rhombus to spindle-shaped same as fibrocyte and the intercellular space is widen, cells are separated from each other.4. Fluorescent quantitation testing shows that the expression of a-SMA mRNA is up-regulation and E-cad mRNA is down-regulation in cultered type II penumonocyte (A549) which have been treated with10mM/LCdCl2for24h,48h and72h.5. Western blot testing shows that with the increase of time exposs to Cd, epithelial specificity maker E-cadhering (E-cad) expression is wind-down and mesenchymal specificity maker a-SMA expression is increased gradually in cultured type Ⅱ penumonocyte cell.6. Western blot testing shows following with the increase of time exposed to Cd, p-EGFR is increased gradually, which represents the activation status of EGFR signaling pathway. Conclusion1. Both animal experiment and dead case of cadmium poisoning support the view that cadmium exposure can induce lung fibration, and in the group exposed to CdCl2through respiratory is more grievous.2. Animal experiment indicates that Cd can induce type Ⅱ penumonocyte translation into fibrolast.3. In vitro cell culture experiment indicates that Cd can induce the type Ⅱ penumonocyte translation into fibrolast.4. In the process of EMT epithelial specificity maker E-cadhering (E-cad) expression is wind-down and mesenchymal specificity maker a-SMA expression is increased gradually.5. All of the results support the view that EMT plays an important part in the process of cadmium-induced pulmonary fibrosis.6. The activation of EGFR signal transduction pathway is related to to cadmium induced EMT and lung fibration.更多还原