【作者】 程洁；

【导师】 洪法水；

【作者基本信息】 苏州大学 ， 生物物理， 2013， 硕士

【摘要】 随着稀土应用的日趋广泛，不可避免地进入环境中并在生物体内累积，其对人体健康可能造成的影响己引起人们的普遍关注。作为轻稀土元素最重要的蓄积器官，稀土对肝脏的损伤作用值得深入探讨。目前，国内外已开展稀土元素对肝毒性和免疫功能影响的研究，但其分子机制尚未清楚。鉴于此，本论文主要研究稀土长期摄入对肝脏及机体免疫功能的影响，为制定稀土使用安全标准和评价稀土生物效应提供实验依据。论文主要涉及以下内容：（1）镧系元素（Ln）可以对大鼠和小鼠产生各种毒性作用，但是其作用机制仍不明朗。本实验中，用等离子电感耦合-质谱、不同光谱方法、凝胶电泳及透射电子显微镜的技术手段，以不同剂量（2、10和20mg/kg BW）的CeCl₃持续灌胃小鼠45天观察肝脏DNA构象变化及肝细胞凋亡程度。结果表明CeCl₃处理后小鼠肝体比显著上升。肝脏中铈元素明显累积，并插入到DNA碱基对中或与DNA核苷酸结合，从而改变DNA构象。此外，通过凝胶电泳及透射电子显微镜观察，高剂量的Ce³⁺可导致DNA断裂和肝细胞凋亡。（2）已有的研究证实Ln可引起小鼠肝损伤，然而其引发凋亡的分子机制仍不清楚。本实验以不同剂量（2、10和20mg/kg BW）的CeCl₃持续灌胃小鼠60天，分析肝细胞超微结构变化、氧化胁迫水平、多种氧化应激物、酶以及与应激有关的基因表达水平的变化情况。研究发现，CeCl₃暴露60天后，肝脏中铈元素明显累积，从而引发肝细胞凋亡。CeCl₃显著促进活性氧（ROS）的产生，并抑制应激相关基因（超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶、金属硫蛋白、热休克蛋白70、谷胱甘肽-硫-转移酶、P53和铁转运蛋白）的表达水平；同时有效地激活细胞色素p4501A的表达。这些结果表明CeCl₃可以导致小鼠肝细胞凋亡，并改变与金属排毒/代谢调节和自由基清除有关基因的表达水平。（3）Ln在肝中累积，并造成一系列损伤，但对Ln造成的肝损伤与基因表达谱的关系研究却知之甚少。以2mg/kg BW剂量CeCl₃连续灌胃处理雄性小鼠90天，结合基因芯片技术，分析肝损伤及基因表达的变化情况。结果发现长期暴露后导致Ce元素在肝中累积，引发肝炎症反应和肝细胞坏死。同时，Ce累积使白细胞、淋巴细胞、血小板、网织红细胞（Ret）和嗜中性粒细胞及A/G比率明显下降；而碱性磷酸酶、乳酸脱氢酶和胆碱酯酶活性及甘油三酯和总胆固醇的浓度显著上升。基因芯片数据显示有1131个基因差异表达，其中，636个基因显著性上调，其余495个基因显著性下调。675个已知功能基因的表达变化与免疫/炎症反应、细胞凋亡、氧化应激、代谢过程、细胞周期、细胞增殖、细胞骨架、信号传导、转录、翻译和运输有关。尤其CeCl₃长期暴露后Nt5e的表达水平显著下调，引发肝炎症反应，而Cyp4a12a的过表达和Cdkn1a的低表达导致肝代谢紊乱。（4）已有研究证明Ln可以导致小鼠脾细胞凋亡，并降低其免疫能，但其分子机制仍不明朗。为研究Ln暴露造成小鼠脾凋亡的分子机制，本实验以不同剂量的（2、10和20mg/kg BW）CeCl₃连续灌胃处理60天，分析Ce累积、脾细胞凋亡及凋亡相关细胞因基因和蛋白的表达情况。结果发现长期CeCl₃暴露后小鼠脾脏中Ce元素明显累积，导致脾系数显著上升和脾细胞凋亡。而且，CeCl₃显著激活caspase-3和caspase-9，抑制Bcl-2的基因和蛋白表达水平，促进活性氧（ROS）的产生。说明CeCl₃是通过内在途径诱导脾细胞凋亡。更多还原

【Abstract】 With the increasingly wide application of lanthanides （Ln）, interests in thepotential benefits of Ln and a greater production of the material have already led to anincreased concern about the potential toxic effects resulting from their uses orunintentional releases into the environment. As the the most important accumulationorgan of light rare earths, it is important to research the effects on liver injury afterexposure to rare earths. Lately, numerous studies have reported that Ln could causebiological and toxic effects of liver. But the mechanisms of these effects need to befurther studied. In view of this, we investigated the molecular mechanisms of liverdamage and immune function changes in mice, to make a comprehensive evaluation onthe effects of Ln and provide some scientific data for protection of peoples＇health andreasonable utilization of Ln. The main results are listed as follows:（1） Ln were shown to cause various toxic effects both in rats and mice; however,the molecular mechanism by which Ce exert theirs toxicity is still understood. In thissection, the impairment of liver DNA conformation and liver apoptosis of mice causedby2,10and20mg/kg BW CeCl₃for consecutive45days was studied in vivo usinginductively coupled plasma-mass spectrometry, various spectral methods, gelelectrophoresis, and transmission electron micrograph. We found that the coefficients ofliver to body weight of the mice treated with CeCl₃were significantly increased. Ce3+could be significantly accumulated in the liver, and it inserts itself into DNA base pairsand/or bind to DNA nucleotide, and alter the conformation of DNA. Furthermore, theevaluation by gel electrophoresis and transmission electron micrograph showed thathigher dose of Ce3+could cause DNA cleavage and hepatocyte apoptosis in mice.（2） Ln has been demonstrated to damage liver of mice, but very little is knownabout the molecular mechanism underlying the mouse liver apoptosis. In order tounderstand the liver injury induced by intragastric administration of CeCl₃（2,10and20 mg/kg BW） for consecutive60days, the hepatocyte ultrastructure, various oxidativestress parameters, and the stress-related gene expression levels were investigated for themouse liver. The results demonstrated that CeCl₃had an obvious accumulation in themouse liver, leading to a classical hepatocyte apoptosis. CeCl₃significantly promotedthe accumulation of reactive oxygen species and inhibited the stress-related geneexpression of superoxide dismutase, catalase, glutathione peroxidase, metallothionein,heat-shock protein70, glutathione-S-transferase, P53, and transferrin, and it effectivelyactivated the cytochrome p4501A. It implied that CeCl₃resulted in apoptosis andalteration of expression levels of the genes related with metal detoxification/metabolismregulation and radical scavenging action in mice.（3） Numerous studies have demonstrated Ln accumulation in the liver, and thecorresponding damage; however, very little work has been done to evaluate therelationship between Ln-induced liver injury and its gene expression profile in mice. Inthis study, liver injury and gene-expressed profiles in male mice induced by oraladministration of CeCl₃（2mg/kg BW） via gavage for consecutive90days wereinvestigated. The results showed that cerium accumulation, liver inflammation, andhepatocyte necrosis were observed. CeCl₃exposure significantly decreased the countsof white blood cells, lymphocyte, and platelet, the reticulocyte count （Ret） andneutrophilic granulocyte percentages as well as A/G ratio, whereas markedly increasedthe activities of alkaline phosphatase, lactate dehydrogenase, and cholinesterase, and theconcentrations of triglycerides and total cholesterol. Furthermore, microarray results ofliver showed that1131genes were counted as differently expressed after long-termexposure to CeCl₃in mice liver. Of these genes,636were upregulated and495weredownregulated. The differential expression of675known function genes involved inimmune/inflammation response, apoptosis, oxidative stress, metabolic process, cellcycle, cell proliferation, cytoskeleton, signal transduction, transcription, translation, andtransportation in CeCl₃exposed livers, respectively. Specifically, the significantdownregulation of Nt5e led to inflammation, overexpressed Cyp4a12a and greatsuppression of Cdkn1a resulted in hepatocyte apoptosis, marked elevation of Cel, andCyp7b1expression caused the metabolic disorders in mouse liver after long-term CeCl₃exposure. Therefore, these genes may be in great relation to liver damages induced byexposure to CeCl₃.（4） Ln has been demonstrated to cause spleen apoptosis and decreased immunity of mice, but very little is known about the molecular aspects of these mechanisms. In orderto understand the spleen apoptotic mechanism induced by intragastric administration of2,10and20mg/kg BW CeCl₃for consecutive60days, we investigated the ceriumaccumulation, apoptosis, the expression levels of the apoptosis-related cytokines genesand proteins. The results demonstrated that cerium had obvious accumulation in mousespleen, leading to the significant increase of the spleen indices and splenocyte apoptosis.Furthermore, CeCl₃could effectively activate caspase-3and-9, decrease the Bcl-2levels of gene and protein, and increase the levels of Bax, and cytochrome C genes andtheir protein expression, and promote reactive oxygen species production. It impliedCeCl₃-induced apoptosis in the mouse spleen via intrinsic pathway.更多还原