2,2’,4,4’-四溴联苯醚(BDE-47)对人胚肾细胞(HEK293)的毒理效应及作用机制研究

	2,2’,4,4’-四溴联苯醚(BDE-47)对人胚肾细胞(HEK293)的毒理效应及作用机制研究
	曹璐璐
学位类型	硕士
导师	吴惠丰
	2015-02
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	环境科学
关键词	2 2’ 4 4’-四溴联苯醚人胚肾细胞毒理效应毒理效应机制细胞凋亡
摘要	多溴联苯醚(PBDEs)是一类典型的溴系阻燃剂，其具备良好的阻燃性及热稳定性，被广泛应用于日常生活用品中。然而，在这些产品的生产及使用过程中，PBDEs极易扩散，并可长期存在于各类环境介质中。此外，其亲脂性特征可使其在生物体中蓄积，并通过食物链最终富集至人体，对机体健康造成不利影响。已有研究表明，PBDEs对人体存在多种毒理效应，包括神经毒性、肝脏毒性及内分泌干扰作用等。肾脏是重要的排泄器官，其细胞损伤很可能影响肾脏功能的正常发挥，因此，亟需开展有关PBDEs对肾细胞的毒理效应及其效应机制的研究。本研究选取PBDEs主要系列物——2,2’,4,4’-四溴联苯醚(BDE-47)作为研究对象，以人胚肾细胞HEK293作为受试细胞系，利用光谱法、流式细胞术、实时荧光定量PCR、蛋白免疫印迹及代谢组学等技术手段，全面考察了BDE-47对人HEK293细胞的毒理效应，并阐明了其毒理效应机制，为PBDEs的毒理学研究及其环境风险评价提供理论依据。本论文研究结果如下： (1). 一定剂量BDE-47 (10^-6～10^-4 M)对HEK293细胞的毒理效应分为毒性响应及代谢响应两部分。毒性响应结果显示：10^-6～10^-4 M BDE-47可诱导细胞产生毒物兴奋效应；当BDE-47浓度为10^-5 M时，可同时造成细胞内ROS水平升高及细胞凋亡现象，指示两者可能具有相关性。代谢响应结果显示：10^-6～10^-4 M BDE-47可造成HEK293细胞内多种代谢物变化，且存在剂量-效应关系；分析表明，BDE-47可引起HEK293细胞的能量代谢紊乱、蛋白质分解代谢增加、胞内氧化应激状态及细胞膜的压力响应等现象。 (2). 一定剂量BDE-47 (10^-6～10^-4 M)对HEK293细胞的毒理效应机制体现于转录水平及翻译水平，研究主要分为以下三部分。 a. 首先，由HEK293细胞中凋亡相关基因的表达水平变化情况可知，BDE-47对基因表达水平的影响集中于10^-5～10^-4 M，可造成p53、Bcl-2家族成员的编码基因(Bcl-2、Bax、Bad及Hrk)及Caspases家族成员的编码基因(Caspase-8)的表达水平上调。研究结果表明：BDE-47可通过外源死亡受体途径及内源线粒体途径诱导凋亡。其中，p53及Bcl-2家族主要通过内源线粒体途径发挥调节作用；Caspase-8则可同时参与死亡受体途径及线粒体途径；在凋亡最后的执行阶段，HEK293细胞可能通过非Caspases依赖的途径实现细胞凋亡。 b. 其次，由HEK293细胞中细胞增殖(SFRS3)、氧化应激(APE1)及凋亡(p53)相关蛋白的表达水平变化情况可知，随BDE-47浓度的变化，SFRS3及APE1蛋白表达水平分别与细胞相对增殖率及胞内ROS水平存在一致性。实验结果说明，SFRS3与BDE-47诱导的HEK293细胞增殖变化密切相关；APE1可能是细胞氧化应激与细胞凋亡间重要的中介因子，可通过诱导p53的高表达引起凋亡。 c. 最后，由p53启动子区域基因片段(p53-DNA)的光谱变化情况可知，10^-6～10^-5 M BDE-47可造成p53-DNA紫外-可见吸收光谱产生增色效应，并使EB-p53-DNA体系发生静态荧光猝灭，此外，10^-5 M BDE-47可使p53-DNA的熔点上升0.5 ^oC。综合以上结果推测，BDE-47主要以沟槽形式与p53基因结合，并造成其损伤。
其他摘要	Because of good flame retardancy and thermal stability, polybrominated diphenyl ethers (PBDEs) are extensively used as brominated flame retardants in products of daily life. However, PBDEs can release from products easily and persist in various environmental media for a long time. Furthermore, high liposolubility contributes to accumulation of PBDEs in organisms, and subsequent biomagnification allows PBDEs to be transferred to human through food chain and exert adverse effects on human health. Previous studies have revealed that PBDEs could cause various toxic effects, including developmental neurotoxicity, hepatotoxicity and endocrine disruption. Kidney is an important excretive organ, and renal cell injury may influence renal function. Therefore, it is necessary to investigate toxic effects and toxicity mechanism of PBDEs on kidney cell lines. In the present study, one of predominant PBDE congeners in human body - 2,2’,4,4’-tetra-bromodiphenyl ether (BDE-47) was chosen to study its toxic effects and mechanism in human embryonic kidney 293 cells (HEK293). Multiple techniques were applied in this study, such as spectroscopy, flow cytometry, quantitative real-time PCR (qRT-PCR), western blotting and NMR-based metabolomics, etc. The results were supposed to provide scientific evidences for toxicological study and ecological risk assessment of PBDEs. The major results were summarized as follows. (1). Toxic effects were induced by the concentration range of BDE-47 (10^-6- 10^-4M) on HEK293 cells, including toxic and metabolic responses. The results of toxic responses showed that BDE-47 caused hormesis effect in HEK293 cells from 10^-6to 10^-4M^{. Cell apoptosis and ROS overproduction detected}at 10^{-5 M of BDE-47 indicated the correlation between them.}Metabolic response revealed that BDE-47 induced dose-responsive effects on metabolite variations of HEK293 cells at selected concentrations (10^-6- 10^{-4 M}). In details, the metabolic responses indicated the disturbance in energy metabolism, stimulation of protein catabolism, intracellular oxidative stress status and stress-response of cellular membrane caused by BDE-47 exposures in HEK293 cells. (2). Toxic mechanism of HEK293 cells induced by the concentration range of BDE-47 (10^-6- 10^-4M) were studied at transcription and translation levels. a. Firstly, the expression levels of apoptosis-related genes suggested that BDE-47 could raise the expression levels of p53, Bcl-2 family-encoding genes (Bcl-2, Bax, Bad and Hrk) and Caspases family-encoding genes (Caspase-8) at concentrations of 10^-5and 10^-4M. The results revealed that BDE-47 caused HEK293 cells apoptosis through death receptor and mitochondrial pathways. p53 and Bcl-2 family mainly played regulatory roles in mitochondrial pathway. Caspase-8 could be involved in both death receptor pathway and mitochondrial pathway. In the executional stage, HEK293 cells apoptosis might be induced by Caspases-independent pathway. b. Secondly, the expression levels of proteins related to cell proliferation (SFRS3), oxidative stress (APE1) and cell apoptosis (p53) suggested there was consistency between SFRS3 protein expression and cell proliferation ratio as the concentration of BDE-47 increased. The expression level of APE1 protein was also consistent with the intracellular ROS level. The results revealed that SFRS3 was closely associated with the alteration of HEK293 cell proliferation caused by BDE-47. APE1 might be an important mediator between oxidative stress and cell apoptosis by inducing high expression of p53. c. Lastly, the spectral changes of the promoter region segment of the p53 gene (p53-DNA) showed that as the concentration of BDE-47 increased (10^-6- 10^-5 M), p53-DNA produced a hyperchromic effect and the fluorescence system of EB-p53-DNA had a static quenching. Furthermore, the melting point of p53-DNA increased by 0.5 ^oC in the presence of 10^-5M of BDE-47. Spectroscopic results suggested that groove binding was the major binding mode of BDE-47 with p53 gene causing DNA damage.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/7926
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	曹璐璐. 2,2’,4,4’-四溴联苯醚(BDE-47)对人胚肾细胞(HEK293)的毒理效应及作用机制研究[D]. 北京. 中国科学院大学,2015.

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