【作者】 郭志英；

【导师】 苏旭；

【作者基本信息】 中国疾病预防控制中心 ， 卫生毒理学， 2009， 博士

【摘要】 氡(Radon)是一种广泛存在于环境空气中的天然放射性惰性气体,在正常本底地区,人类所受到的天然辐射中,氡及其子体所致的剂量最大,占全部天然辐射剂量的50%,在一些地下矿井及使用含氡建筑材料装饰的居室内可以出现更高的氡水平。由于氡及其子体的特殊作用方式,支气管上皮基底细胞、粘液细胞及肺上皮细胞成为其重要的作用靶点,其衰变产生的α粒子会对靶细胞产生较强的生物学效应。吸入氡及其子体对人体的主要危害是引发肺癌,人类肺癌中有10%左右归因于氡及其子体的照射。国际癌症研究机构(IARC)已经将氡及其子体划归为I类致癌因素。近年来,随着人们对氡及其子体危害认识的进一步加深,关于氡及其子体的生物效应、作用机制及防护对策的研究,逐渐成为全球范围内预防医学、公共卫生和环境保护界的研究热点。在本研究中,我们首先建立了氡及其子体吸入染毒的小鼠动物模型。在不同染毒剂量和不同时间点动态观察染毒小鼠肺及支气管的病理损伤。氡吸入染毒后24h内,小鼠肺组织中出现不同程度的充血、出血、气肿、水肿、渗出以及炎症细胞浸润等急性呼吸道损伤和炎症的表现。随着染毒剂量的增加,各种类型病理变化的发生比例、范围和严重程度有增加的趋势。染毒结束后,随着饲养时间的延长,水肿和增生的发生比例呈现先增高后降低的趋势,由早期以肺泡上皮细胞增生为主,逐渐演变为以肺间质细胞增生为主。肺泡毛细血管内血栓的发生率逐渐增高;染毒后90d出现了肺泡的萎缩和纤维化。整个实验中未见细胞的异型性改变。实验表明,氡及其子体沉积于呼吸道后,会引起肺及支气管组织的进行性损伤,其病理变化是外界因素(氡及其子体)和机体共同作用的结果,表现为急性炎症、损伤,肺上皮细胞和成纤维细胞增生及肺组织纤维化三个阶段的动态发展的过程,该过程与放射性肺损伤的病理变化相似。增生和纤维化是机体应对损伤性刺激的重要反应,过度的增生和纤维化可能是肿瘤发生的原因之一。氡及其子体在诱发肺及支气管损伤的过程中,可能涉及到众多相关基因的改变及功能的异常,这些基因的激活或抑制,协同或拮抗,将会影响病变的进程。本研究中分别以吸入染毒后不同时间段(<24h和90d)的小鼠为研究对象,采用抑制性差减杂交技术(SSH),分别构建了氡染毒后两个时间段的小鼠肺及支气管组织差异表达基因的cDNA文库。将差异表达的cDNA片段测序,结合同源性检索和生物信息学分析,对差异表达基因进行初步功能分类。扣除重复克隆,染毒后24h内筛选组中有89个阳性克隆,染毒后90d组中有53个阳性克隆中插入的cDNA片段序列与已知序列有不同程度的同源性。这些序列主要编码以下几类蛋白:1.细胞因子及其受体相关蛋白;2.激酶及其相关蛋白;3.其他酶类;4.一些未知功能的蛋白或是从核酸序列推算出来的假想蛋白;5.其他还包括细胞骨架蛋白、纤维连接蛋白、通道蛋白、热休克蛋白、癌基因和抑癌基因的编码产物等。筛选得到的基因中,有的可能参与细胞凋亡、周期调控、组织纤维化、免疫调节、细胞代谢及细胞间信号转导等过程。氡染毒小鼠肺及支气管组织差异表达基因的获得,为进一步研究氡致肺损伤及演变为肺癌的分子生物学机制提供了实验依据。综合文献调研,在筛选得到的基因文库基础上,我们选择TGF-β、MAPK信号通路成员及Akt基因,利用分子生物学的相关实验技术,初步研究它们在氡吸入染毒不同剂量和不同阶段小鼠肺组织中的表达情况。TGF-β是目前公认的与放射性肺纤维化发生密切相关的细胞因子,它能促进成纤维细胞合成胶原纤维。MAPKs是存在于大多数细胞内的一系列丝/苏氨酸蛋白激酶,该通路激活后可参与细胞生长、发育及细胞间功能同步等生理功能,它主要包括JNK/SAPK、P38MAPK和ERK三条生化级联途径。Akt信号通路是体内的重要的生存信号通路,在人类的多种恶性肿瘤中均有p-Akt表达,p-Akt蛋白的水平与肿瘤的预后有关。染毒后24h内,小鼠肺组织中TGF-β的蛋白表达水平有随着染毒剂量的增加而增加的趋势,染毒后随着时间的推移,TGF-β蛋白表达水平逐渐增高(30、90和180d组的p<0.05)。染毒后24h内,小鼠肺及支气管组织中JNK mRNA和蛋白表达水平随着染毒剂量的增加而增高,JNK蛋白磷酸化水平增高明显(30、50和60WLM组的p<0.01);染毒后随着时间的推移,JNK mRNA和蛋白表达量呈现先升高后降低的特征,染毒后24h内达到最高,JNK蛋白的磷酸化水平也呈现先升高后降低的趋势,在染毒后30d达到峰值(p<0.01)。染毒后小鼠肺组织中的p38 MAPK蛋白及其磷酸化水平随着染毒剂量的增加而增高,其中染毒60 WLM组样本的p-p38蛋白表达增高明显(p<0.01);氡染毒后随着时间的延长,小鼠肺组织中的p38MAPK蛋白及其磷酸化水平呈现先增高后降低的趋势,在染毒30d时达到最高。在不同染毒剂量和染毒后不同时间段,小鼠肺及支气管组织中ERK和p-ERK蛋白的表达量与对照组无明显差异。染毒后小鼠肺组织中的p-Akt蛋白表达水平随着染毒剂量的增加而增高,高磷酸化水平维持较长的时间。本研究表明,与肺组织受到γ射线照射后的变化相似,小鼠吸入氡及其子体后,肺及支气管组织中的TGF-β蛋白表达增强,并且与氡染毒后肺组织增生和纤维化的过程存在时程上的重合,说明TGF-β可能参与了氡染毒后小鼠肺组织发生纤维化的过程。作为一种放射性刺激因素,氡及其子体暴露引起了小鼠肺组织中JNK的表达增强和磷酸化激活,JNK的表达和激活水平与氡染毒的剂量及染毒后持续的时间有关,因此JNK及JNK蛋白的磷酸化水平有可能作为早期判断氡暴露剂量的生物学指标之一。氡及其子体吸入染毒使p38蛋白激活所需的阈值较高,即其敏感性较差,但是当染毒剂量一旦达到阈值时,其激活又是非常强烈的。ERK信号通路未参与氡染毒致肺损伤的过程。Akt蛋白磷酸化水平与染毒剂量成正相关,并且高磷酸化水平维持较长时间,提示染毒小鼠肺组织的病理改变有可能向着恶性发展,预后可能不良。TGF-β与MAPK家族的JNK和p38之间可能存在一定的关联,TGF-β可能通过JNK和p38信号通路将信号传递至细胞核内,从而实现对细胞生长、分化和凋亡的调控;在特定的刺激条件下,MAPK信号通路与Akt信号通路可以被先后激活;同时细胞内可能还存在其他激活MAPK通路的机制,这些过程也有可能是同时存在的。对于TGF-β、MAPK信号通路和Akt信号通路在氡染毒小鼠肺损伤过程中的相互作用机制还有待于进一步深入探讨。更多还原

【Abstract】 Radon is an inert radioactive gas that exists naturally in the air. In normal background areas, the radioactive dose from radon and its decay products is the main natual dose received by mankind, which account for 50% of the total annual absorbed dose. The levels of radon may be higher in some mines and buildings with the new type building materials. Because of the special characteristics of radon and radon progeny, the basal and secretory cells are the primary target of them. Radon will decay into a series of radon decay products, and the alpha particle emitted from radon progeny in the human respiratory will damage the target cells. Causal association of exposure to radon and lung cancer has been demonstrated in epidemiological studies performed on cohorts of miners. Domestic radon has been identified as the most important environmental risk factor for lung cancer. It was reported that 10% of all lung cancer in general populations could be because of indoor radon. International Agency for Research on Cancer (IARC) have recognized radon as a group I carcinogen. In recent years, the potential hazards posed by exposure to alpha radiation from air radon have been of great concern worldwide. And studies of the mechanisms and the protection from radon and its daughters have been become the focus.In the present study, we firstly established an animal model of radon-exposure to study the pathologies of lung and bronchus of the radon-exposure mice at different time points and different dose groups. The radon-exposure caused various pathologies of lung and bronchus, including hyperemia, hemorrhage, emphysema, edema, exudation and inflammation. However, the degeneration, necrosis and loss of lung epithelial cells were not obvious. There was not significant difference between the two dose groups. After radon-exposure, along with the time, the incidence rates of edema and hyperplasia increased and decreased later. The capillary thrombosis became serious. After 90 days, the shrinks and fibrosis of alveolar occurred. No cancer cells were founded in the study. This study also showed that radon and its daughters absorbed in the respiratory tract would damage on lung and bronchus. The pathological changes in lung were dynamic process, which were caused by the interaction between the external factors (radon and its daughters) and the internal factor of the body. After radon-exposure, the lung and bronchus would experience early acute inflammation, proliferation and pulmonary fibrosis three stages. The pathological procedure of lung after radon-exposure was similar to that induced by the other radiation. Hyperplasia and fibrosis were important responses of the body to the external hazardous. The excessive hyperplasia and fibrosis may be one of the inducements of cancer.During the pathological procedure of lung after radon-exposure, a large number of related genes may be involved in. In the SSH test, two pairs of radon-exposure mice of different stages (<24h and 90 days after exposure with 30 WLM) were used as animal models. The control and the treatment groups acted as testers and drivers for the each other. The differentially expressed cDNA libraries in lung and bronchus of mice exposed to radon were constructed. The obtained forward and reverse cDNA fragments were directly inserted into pGEM-T-easy vector and transformed into E. coli DH5α. The inserts in plasmid were amplified by nested polymerase chain reaction (PCR), and the differentially expressed fragments were sequenced. In the end these sequences were aligned with GeneBank data and the differentially expressed genes were classified based on their function. Finally, in the two screening group (<24h and 90 days post-exposure), there were 89 and 53 ESTs respectively were found to be identified with known genes encoding protein of below: Cytokines and receptor-associated protein; Kinases and related proteins; Other proteins including enzymes, heat shock protein, cytoskeleton proteins, fibronectins, products of oncogene, some hypothetical proteins. Those proteins may be involved in the procedures of apoptosis, cell cycle regulation, tissue fibrosis and intracellular signal transduction.Based on the screening constructed libraries, we took TGF-β, members of MAPK signaling pathway and Akt as the target genes for the further study. We use molecular biology techniques to study the expression of them in the lung and bronchus of radon-exposure mice preliminarily. Within 24h post-exposure, TGF-βprotein in lung and bronchus increased along with the increase of expose dose. After radon-exposure with 60 WLM, TGF-βprotein in lung was higher than the control group, and increased with time-lapse. Less than 24 hours post-exposure, the levels of JNK mRNA, JNK protein and the phosphorylation levels of JNK protein in lung all increased with the dose-increasing. After radon-exposure the JNK mRNA, JNK protein and the phosphorylated JNK protein increased first and reduced later. Within 24 hours after exposure, the expression of p38/MAPK protein in each dose group enhanced than the control group. But the increasing level is lower than the JNK protein. The phosphorylated p38/MAPK protein also increased significantly in the samples exposed to 60 WLM. After exposure, the phosphorylated p38/MAPK protein increased first and reduced later. In different doses and stages after radon-exposure, there was not significant difference in the levels of ERK and p-ERK protein compared with the control group. Within 24 hours after exposure, the levels of Akt protein were stable in different dose groups. However, the phosphorylated Akt protein has increased with the dose increasing. After exposure, the Akt and phosphorylated Akt proteins have increased. The phosphorylated Akt protein increased significantly and maintained for a long time.TGF-βexerts its effects on cell proliferation, differentiation and migration partly through its modulation of extracellular matrix components, such as fibronectin and plasminogen activator inhibitor-1 (PAI-1). TGF-βplays an important role in the radiation-induced pulmonary fibrosis formation, and promotes the synthesis of collagen fibers. After radon-exposure, the regularity of TGF-βprotein expression increased simultaneously with the appearance of pulmonary hyperplasia and fibrosis, indicating that TGF-βplays an important role in the procedure of radon-induced pulmonary fibrosis. The JNK signal pathway can be stimulated by various stresses and took part in cellular growth, development, division, differentiation and apoptosis. As one kind of radioactive factor, radon and its daughters can stimulated the transcription, translation and phosphorylation of JNK protein. The levels of JNK and phosphorylated JNK protein have related with the dose and the time after the radon-exposure. The p38 MAPK signaling pathway plays an important role in inflammation, stress reaction, apoptosis and cell cycle regulation. This study showed that the threshold of p38 activation induced by radon-exposure is relatively high and resistant to the stimulation. Once the dose reaches the threshold, the p38 protein was activated dramatically. The ERK signaling pathway is not involved in radon-induced lung injury process. The activation of Akt protein is closely related to the occurrence and development of tumors. A variety of growth factors, hormones, cytokines, and deletion of PTEN gene, as well as activation of Ras could stimulate the activation of Akt. In this study, phosphorylation level of Akt protein was positively correlated with the dose, and maintained at high levels for a long time, suggesting that pathological changes in lung tissue may be aggravated.TGF-βhas relationship with the members of MAPK family, such as JNK and p38. TGF-βcould transfer signalling through the JNK and p38 signal transduction pathway to the nucleus, and regulate the cell growth, differentiation and apoptosis. In some conditions the MAPK and Akt signaling pathways can be activated sequencely. There are other determinants to activate MAPK cascade at the same time. They need further studies to explore the roles of TGF-β, MAPK cascades and Akt in the pathologies induced by radon-exposure.更多还原