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题名:
镉和砷对不同发育阶段紫贻贝毒理效应的组学研究
作者: 于德良
学位类别: 博士
答辩日期: 2017-05-18
授予单位: 中国科学院大学
授予地点: 北京
导师: 吴惠丰 ; 赵建民
关键词: 紫贻贝 ; ; ; 毒理效应 ; 代谢组学 ; 蛋白质组学
学科分类: 环境科学技术::环境科学技术基础学科::环境生态学 ; 环境科学技术::环境科学技术基础学科::环境毒理学
学位专业: 环境科学
中文摘要: 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。
英文摘要: 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
内容类型: 学位论文
URI标识: http://ir.yic.ac.cn/handle/133337/22011
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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

Recommended Citation:
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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