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题名	镉和砷对褐牙鲆幼鱼毒理效应的组学研究
作者	刘佳琳
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
关键词	重金属，镉，砷，毒理效应，褐牙鲆
中文摘要	本研究根据目前我国渤海海岸带重金属污染现状，选择典型重金属镉、砷(V)为暴露污染物，以近海重要渔业生物褐牙鲆（Paralichthys olivaceus）为研究对象，采用代谢组学、蛋白质组学及其它分子生物学技术手段，探讨镉、砷污染物对褐牙鲆的毒理效应机制，研究结果有助于为重金属的环境风险评估提供一定的理论依据。论文获得如下结果： 1. 筛选了基于核磁共振波谱技术的褐牙鲆幼鱼组织代谢组学数据预处理方法       以褐牙鲆（Paralichthys olivaceus）幼鱼为研究对象，利用核磁共振技术，研究了褐牙鲆幼鱼全组织在低盐胁迫48小时后的代谢组变化，比较了三种不同预处理方法（单位方差换算、帕莱托换算与对数化-中心化处理）对于低盐胁迫诱导的代谢响应分析的差异。结果表明，褐牙鲆幼鱼体内的渗透调节物-氮氧三甲胺浓度是其它代谢物浓度的200倍以上，低盐胁迫可诱导褐牙鲆幼鱼体内显著的代谢响应。其中，对数化-中心化处理的代谢组数据，经正交偏最小二乘判别分析后，能发现8个低盐胁迫诱导的差异代谢物（不含1个未知代谢物），与代谢物定量结果一致，多于其他两种预处理方法（单位方差换算、帕莱托换算）的代谢组分析结果，对数化-中心化处理的代谢组学预处理方法适合用于褐牙鲆代谢组学分析。研究结果为褐牙鲆代谢组数据的精确分析提供了方法学依据。但本方法是否适用于其他海洋生物代谢组数据预处理，仍需要进一步验证。 2. 镉对褐牙鲆幼鱼毒理效应的研究       褐牙鲆幼鱼分别经低浓度镉（5 µg/L）和高浓度镉（50 µg/L）胁迫14天后，利用双向电泳结合质谱检测技术研究褐牙鲆蛋白质的变化，共获得23个差异蛋白，其中低浓度镉暴露组差异表达的蛋白点共13个，包括2个上调的蛋白点及11个下调的蛋白点；高浓度镉暴露组差异表达的蛋白点共10个，包括9个上调的蛋白点及1个下调的蛋白点。通过对生物学过程进行分析，发现镉胁迫主要干扰了褐牙鲆幼鱼的物质和能量代谢、信号传导和物质转运、细胞骨架构建和防御机制等生物过程。基于核磁共振技术的代谢组学结果表明，镉胁迫主要干扰了褐牙鲆幼鱼蛋白水解能力（丙氨酸、谷氨酸）、渗透调节（牛磺酸）及物质和能量代谢过程（胆碱、磷酸胆碱、葡萄糖、乳酸、腺嘌呤与一磷酸腺苷），特别是大量乳酸的生成表明褐牙鲆幼鱼通过加强厌氧代谢来应对镉胁迫。褐牙鲆特定基因表达差异情况表明，低镉胁迫干扰褐牙鲆幼鱼能量代谢（LDH）和生长发育（IGFBP4）过程；高镉胁迫干扰了褐牙鲆幼鱼性腺分化（SOX9）过程。 3. 砷(V)对褐牙鲆幼鱼毒理效应的研究       褐牙鲆幼鱼分别经低浓度砷（As (Ⅴ), 5 µg/L）和高浓度砷（As (Ⅴ)，50 µg/L）胁迫14天后，利用双向电泳结合质谱检测技术研究褐牙鲆蛋白质的变化，共获得30个差异蛋白，其中低浓度砷暴露组差异表达的蛋白点共15个，包括3个上调的蛋白点及12个下调的蛋白点；高浓度砷暴露组差异表达的蛋白点共15个，包括6个上调的蛋白点及9个下调的蛋白点。通过对生物学过程进行分析，发现砷胁迫主要干扰了褐牙鲆幼鱼的物质和能量代谢、信号传导和物质转运、蛋白折叠和组装、细胞骨架构建等生物过程。基于核磁共振技术的代谢组学结果表明，砷胁迫主要干扰了褐牙鲆幼鱼蛋白水解能力（丙氨酸、谷氨酸、天冬氨酸、甘氨酸、组氨酸、苯丙氨酸）、渗透调节（牛磺酸）及物质和能量代谢过程（胆碱、磷酸胆碱、葡萄糖、糖原、乳酸、延胡索酸、琥珀酸、腺嘌呤与一磷酸腺苷），代谢产物乳酸、糖原、葡萄糖表达上调表明，褐牙鲆幼鱼启动无氧代谢替代有氧代谢来产生ATP。褐牙鲆特定基因表达差异情况表明，低砷胁迫干扰了褐牙鲆幼鱼能量代谢（LDH）过程；高砷胁迫干扰了褐牙鲆幼鱼能量代谢（NADH dehydrogena, MDH）、免疫防御（AMPs, IFN γ）、生长发育（IGFBP4, IGF1）、及抗氧化胁迫（SOD, GSTs）过程。
英文摘要	As environmental contaminants, heavy metals are of great concern due to their high risk to the environment and human health. Cadmium and arsenic are two common heavy metals in the Chinese coastal waters. In this study, the two “-omic” approaches including metabolomics and proteomics were used to elucidate the toxic effects of Cd and As (Ⅴ) on the juvenile flounder Paralichthys olivaceus. The major results were summarized as following:   NMR-based metabolomic data preprocessing method on the extracts of whole tissue from flounder Paralichthys olivaceus       The juvenile flounder Paralichthys olivaceus was exposed to a reduced salinity (50% normal salinity, 15.6 psu). After exposure for 48 h, the one dimensional 1H NMR spectroscopy was used to analyze the metabolomes of flounder samples. To investigate the influences of data preprocessing, the metabolomic data of founder samples were subjected to three different data preprocessing methods (unit variance scaling, Pareto scaling and generalized log transformation-mean centering) before pattern recognition analysis. Results indicated that the concentration of osmolyte, trimethylamine N-oxide, was approximately 200 times higher than other metabolites in flounder samples. The reduced salinity induced significant metabolic responses in juvenile flounder. After orthogonal partial least squares-discriminant analysis (OPLS-DA), generalized log transformation-mean centering was found to be the best data preprocessing method, presenting 8 significantly altered metabolites induced by reduced salinity, which was consistent with the quantitative analysis of metabolites. Overall, this study provided a suitable data preprocessing method to analyze the metabolomic data of flounder P. olivaceus. 2. Omic analyses reveal toxicological effects of Cd on flounder P. olivaceus       The present study aimed at evaluating the toxicological effects of cadmium (5 µg/L and 50 µg/L) in P. olivaceus. After exposure for 14 days, 24 spots in all which were significantly expressed in P. olivaceus with cadmium exposures. These proteins can be categorized into diverse functional classes, related to material and energy etabolism、signal transduction and transport、transcription and translation、production and development、cytoskeleton、apoptosis and defense mechanisms. Metabolic profiling indicated that Cd2+-treatment enhanced proteolysis and disturbed osmotic regulation and energy metabolism in juvenile flounder P. olivaceus. Furthermore, the enhanced anaerobic metabolism indicated by the elevated lactate in flounder tissue extracts. Gene expression profiles exhibited that Cd could induce immune stress and oxidative stress and disturb energy metabolism in juvenile flounder P. olivaceus. In addition, Cd might promote the growth and gonadal differentiation in juvenile flounder P. olivaceus. 3. Omic analyses reveal toxicological effects of As (Ⅴ) on flounder P. olivaceus       The present study aimed at evaluating the toxicological effects of arsenic (As (Ⅴ), 5 µg/L and 50 µg/L) in P. olivaceus. After exposure for 14 days, 30 spots in all which were significantly expressed in P. olivaceus with arsenic exposures. These proteins can be categorized into diverse functional classes, related to material and energy etabolism、signal transduction and transport、transcription and translation、protein folding and assembly and cytoskeleton. Metabolic profiling indicated that As (Ⅴ)-treatment enhanced proteolysis and disturbed osmotic regulation and energy metabolism in juvenile flounder P. olivaceus. Furthermore, the enhanced anaerobic metabolism indicated by the elevated lactate、glucose and glycogen in flounder tissue extracts. Gene expression profiles exhibited that arsenic could induce immune stress and oxidative stress and disturb energy metabolism and promote the growth in juvenile flounder P. olivaceus.
学科主题	环境科学技术其他学科
内容类型	学位论文
源URL	[http://ir.yic.ac.cn/handle/133337/21984]
专题	中科院烟台海岸带研究所知识产出_学位论文 烟台海岸带研究所_支撑部门
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	刘佳琳. 镉和砷对褐牙鲆幼鱼毒理效应的组学研究[D]. 北京. 中国科学院大学. 2017.

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