镉和砷对不同发育阶段紫贻贝毒理效应的组学研究

	镉和砷对不同发育阶段紫贻贝毒理效应的组学研究
	于德良
学位类型	博士
导师	吴惠丰 ; 赵建民
	2017-05-18
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	紫贻贝镉砷毒理效应代谢组学蛋白质组学
摘要	近年来渤海的重金属污染问题日益严重。本论文根据渤海重金属污染现状，利用代谢组学和蛋白质组学技术，并结合传统毒理学方法研究了两种海洋典型重金属污染物镉（Cd）和砷（As）对不同发育阶段紫贻贝（Mytilus galloprovincialis）的毒性效应机制，旨在从整体水平探讨 Cd 和 As 对不同发育阶段紫贻贝胁迫机制的差异，并从中筛选出表征污染物胁迫效应的代谢物及蛋白质作为生物标志物，为海洋环境监测及评价提供理论依据。研究结果如下： 1. 不同浓度 Cd 和 As (V)对紫贻贝 D 型幼虫毒理效应的组学研究紫贻贝 D 型幼虫经不同浓度 Cd（5 μg/L 和 50 μg/L）和 As (V)（5 μg/L 和50 μg/L）暴露 48 小时后，不同浓度的 Cd 和 As (V)暴露对紫贻贝 D 型幼虫均未造成致畸作用。Cd 和 As (V)对 D 型幼虫的致毒机制存在显著差异，其中 Cd 对D 型幼虫在分子水平存在剂量-效应关系。基于核磁共振技术的代谢组学研究结果表明，Cd 和 As (V)暴露均影响了 D 型幼虫能量代谢、脂肪代谢和渗透压调节等生物过程。基于双向电泳结合质谱检测技术的蛋白质组学研究发现，5 μg/L 和50 μg/L Cd 暴露后紫贻贝 D 型幼虫组织中与细胞骨架、能量代谢、基因表达和免疫调节等生物过程有关的蛋白质发生显著变化。5 μg/L Cd 暴露诱导了 D 型幼虫组织中与细胞凋亡、氧化应激和蛋白质折叠相关的蛋白质出现显著变化。50 μg/L Cd 暴露诱导了 D 型幼虫组织中与解毒和蛋白质代谢相关的蛋白质出现显著变化。5 μg/L As (V)暴露干扰了 D 型幼虫的蛋白质代谢等生物过程，50 μg/L As (V)暴露影响了 D 型幼虫的解毒和免疫调节等生物过程。 2. Cd 和 As (V)对紫贻贝稚贝毒理效应的组学研究紫贻贝稚贝经 50 μg/L Cd 和 50 μg/L As (V)暴露 48 小时后，重金属测定结果表明，Cd 暴露组紫贻贝稚贝组织中 Cd 富集量为极显著水平（p < 0.01），而 As (V)暴露组 As 富集不显著。代谢组学研究结果表明，Cd 和 As (V)均干扰了紫贻贝渗透压调节和能量代谢等过程。Cd 影响了稚贝的脂肪代谢过程，支链氨基酸的显著下调表明 As (V)干扰了稚贝的免疫调节过程。蛋白质组学结果发现，Cd暴露干扰了稚贝的细胞骨架、免疫调节和信号转导等过程，而 As (V)暴露影响了稚贝能量代谢、细胞凋亡、基因表达和氧化应激反应等过程。经 Cd 和 As (V)暴露后，紫贻贝稚贝组织中不同抗氧化酶（超氧化物歧化酶、过氧化氢酶和谷胱甘肽转移酶）活性和丙二醛含量均未出现显著变化。结果表明，Cd 和 As (V)暴露对紫贻贝稚贝的致毒机制存在显著差异。 3. Cd 和 As (V)对紫贻贝成贝毒理效应的组学研究紫贻贝成贝暴露于 50 μg/L Cd 和 50 μg/L As (V) 48 小时后发现，Cd 和 As (V)暴露对紫贻贝成贝的致毒机制存在差异。重金属含量测定结果表明，Cd 暴露组成贝组织中 Cd 富集量为极显著水平（p < 0.01），As (V)暴露组成贝组织中 As富集量为显著水平（p < 0.05）。代谢组学研究发现，Cd 暴露和 As (V)暴露均可诱导成贝组织中与渗透压调节和能量代谢相关的代谢物发生显著变化，但相应的胁迫应答机制不同。蛋白质组学研究结果表明，Cd 暴露导致紫贻贝成贝组织中细胞骨架的稳定性受到干扰，而 As (V)暴露影响了成贝组织中基因表达、脂肪代谢和神经传导等生物过程，并诱导了 DNA 损伤。综上，紫贻贝三个发育阶段（D 型幼虫期、稚贝期和成贝期）经重金属 Cd和 As 胁迫后均出现不同代谢物和蛋白质的显著变化，这些代谢物和蛋白质分别对应不同的生物过程。其中，D 型幼虫对 Cd 和 As 暴露反应最为敏感，发生显著变化的代谢物和蛋白质数量最多，涉及到的生物过程最为广泛，并且 D 型幼虫在分子水平对 Cd 胁迫存在剂量-效应关系；其次，稚贝对 Cd 和 As 暴露反应敏感度降低，出现显著变化的代谢物和蛋白质数量减少，但仍有多个生物学过程受到干扰，并影响了稚贝生长；最后，紫贻贝成贝对 Cd 和 As 暴露的耐受度最高，受到影响的生物过程最少。组学研究结果证明，随着紫贻贝生长期的延续，紫贻贝体内器官逐步成熟，其应对 Cd 和 As 胁迫的能力有所增强。
其他摘要	In recent years, the Bohai Sea has been heavily polluted by heavy metals. In this study, we selected two typical heavy metal pollutants, cadmium (Cd) and arsenic (As), for further exposure experiments. Three developmental stages of marine environmental bioindicator, mussel Mytilus galloprovincialis, were chosen for this study. We combined traditional toxicological methods and -omic technologies to investigate the toxicological effects and mechanism of heavy metal pollutants in mussels and characterized the metabolite and protein biomarkers related to the toxicological effects of Cd and As. These biomarkers may be used for marine environmental monitoring. Results are described as follows: 1. Toxicological effects of different concentrations of Cd and As (V) in D-shape larval mussels D-shape larval mussels were exposed to different concentrations of Cd (5 and 50 μg/L) and As (V) (5 and 50 μg/L) for 48 hours. Results indicated that there was no obvious aberration effect in D-shape larval mussels exposed to different concentrations of Cd and As (V). NMR-based metabolomics revealed differential metabolic responses in D-shape larval mussels exposed to Cd and As (V). Basically, both Cd and As (V) caused energy disruption, lipid metabolism and osmotic stress in D-shape larval mussels. In addition, Cd caused a dose-dependent effect in D-shape larval mussels. Proteomic analysis based on 2D electrophoresis and mass spectrometry indicated that both 5 and 50 μg/L of Cd induced cytoskeleton disruption, disturbances in energy metabolism, immune regulation and gene expression in D-shape larval mussels. The low concentration (5 μg/L) of Cd exposure caused disturbances in apoptosis, protein folding and oxidative stress in D-shape larval mussels. The high concentration (50 μg/L) of Cd exposure caused detoxication and protein metabolism in D-shape larval mussels. The low concentration (5 μg/L) of As (V) induced disturbances in protein metabolism, while the high concentration (50 μg/L) of As (V) induced immune stress and detoxication in D-shape larval mussels. 2. Toxicological effects of Cd and As (V) in juvenile mussels After exposed to Cd (50 μg/L) and As (V) (50 μg/L) for 48 hours, juvenile mussels exhibited significant (p < 0.01) Cd accumulations in the whole soft tissues. NMR-based metabolomics revealed differential metabolic responses in juvenile mussels exposed to Cd and As (V). Both Cd and As (V) caused energy disruption and osmotic stress in juvenile mussels. Cd caused disruption of lipid metabolism in juvenile mussels. The decreased branched chain amino acids (BCAAs) suggested the immune stress caused by As (V) exposure in juvenile mussels. Proteomic analysis indicated that Cd caused immune stress and disturbances of cytoskeleton and signal transduction, As (V) induced oxidative disruption and disturbances of energy metabolism, apoptosis and gene expression in juvenile mussels, respectively. There were no significant changes in superoxide dismutase (SOD), catalase (CAT), glutathione S-transferases (GST) activities and malondialdehyde (MDA) content in juvenile mussels. 3. Toxicological effects of Cd and As (V) on adult mussels After exposed to Cd (50 μg/L) and As (V) (50 μg/L) for 48 hours, adult mussels exhibited significant Cd accumulation (p < 0.01) and As accumulation (p < 0.05) in the whole soft tissues. NMR-based metabolomics revealed differential metabolic responses in adult mussels exposed to Cd and As (V). Both Cd and As (V) induced energy disruption and osmotic stress in adult mussels via different pathways. 2D electrophoresis and mass spectrometry-based proteomics revealed that Cd caused cytoskeleton disruption in adult mussels. As (V) induced disturbances of gene expression, lipid metabolism, nerve conduction and DNA damage in adult mussels. In summary, the toxicological effects of Cd and As (V) were characterized in three different life stages (D-shape larvae, juvenile and adult) of marine mussels Mytilus galloprovincialis using traditional toxicological methods and high throughput –omic technologies. Basically, the metabolic and protein responses indicated that the D-shape larval mussel was the most sensitive life stage to the Cd and As (V) exposures. Cd caused a dose-dependent effect in D-shape larval mussels. The reduced sensitivity to Cd and As (V) exposures were observed in juvenile mussels. Adult mussels possessed the highest tolerance to Cd and As stresses. With the growth of mussels, the mussels are more tolerant to Cd and As stresses.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/22011
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	1.中国科学院烟台海岸带研究所 2.中国科学院大学
推荐引用方式 GB/T 7714	于德良. 镉和砷对不同发育阶段紫贻贝毒理效应的组学研究[D]. 北京. 中国科学院大学,2017.

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