氯化铈对肝脏及免疫功能的影响及分子机制研究

发布时间：2018-02-21 07:46

本文关键词： 稀土肝损伤炎症免疫细胞凋亡氧化应激基因表达谱　出处：《苏州大学》2013年硕士论文　论文类型：学位论文

【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CeCl_3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CeCl_3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Ce~(3+)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CeCl_3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，CeCl_3暴露60天后，肝脏中铈元素明显累积，从而引发肝细胞凋亡。CeCl_3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CeCl_3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CeCl_3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CeCl_3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）CeCl_3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CeCl_3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，CeCl_3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CeCl_3是通过内在途径诱导脾细胞凋亡。
[Abstract]:Along with the extensive application of rare earth, inevitably enter the environment and accumulate in the organisms, the potential impacts on human health has aroused widespread concern. As the most important organ in light rare earth elements accumulation, worthy of further discussion on the liver damage effect of rare earth. At present, domestic and foreign research has been carried out on the effects of rare earth elements liver toxicity and immune function, but its molecular mechanism is unclear. In view of this, this paper mainly studies the long-term intake of rare earth effect on liver and immune function, to make use of safety standards and evaluation of rare earth biological effects of rare earth price to provide experimental basis.
The main contents of the paper are as follows:
(1) lanthanide (Ln) can produce a variety of toxic effects on rats and mice, but its mechanism is still not clear. In this experiment, using inductively coupled plasma mass spectrometry, different spectroscopic methods, techniques of gel electrophoresis and transmission electron microscopy, with different doses (2,10 and 20mg/kg BW CeCl_3) for the mice 45 days observation of liver DNA conformation and liver cell apoptosis degree. The results showed that CeCl_3 treated mice liver body ratio increased significantly. Ce3could be significantly accumulated in the liver, and inserted into the DNA base pairs or in combination with DNA nucleotides, thus changing the conformation of DNA. In addition, by gel electrophoresis and transmission electron microscope observation of high dose of Ce~ (3+) could cause DNA cleavage and apoptosis.
(2) research has confirmed that Ln can cause liver damage in mice, but the molecular mechanism of apoptosis remains unclear. In this experiment, different doses (2,10 and 20mg/kg BW) CeCl_3 continued to mice for 60 days, analysis of liver cell ultrastructure changes, oxidative stress level, a variety of oxidative stress, and enzyme changes in the levels of expression and stress related genes. The study found that CeCl_3 60 days of exposure, ce3could be significantly accumulated in the liver, causing liver cell apoptosis of.CeCl_3 significantly promoted the reactive oxygen species (ROS) production, and inhibit the stress related genes (superoxide dismutase, catalase, glutathione peroxidase, metallothionein that heat shock protein 70, glutathione-S-transferase, P53 and iron transport protein) expression level; at the same time effectively activate the expression of cytochrome p4501A. These results indicated that CeCl_3 could induce apoptosis of liver cells in mice, It also changes the expression level of genes related to metal detoxification / metabolic regulation and free radical scavenging.
(3) Ln in the liver and cause a series of damage accumulation, but liver damage and gene of Ln caused by expression of the relationship between spectrum is poorly understood. In 2mg/kg BW dose of CeCl_3 orally treated male mice for 90 days, combined with the technology of gene chip, analysis of hepatic injury and expression of genes. Results showed that after long-term exposure to Ce element accumulation in liver, hepatitis caused by inflammation and necrosis of liver cells. At the same time, the accumulation of Ce lymphocytes, white blood cells, platelets, reticulocyte (Ret) and neutrophils and the ratio of A/G decreased significantly; and alkaline phosphatase, significantly increased the concentration of lactate dehydrogenase and cholinesterase activity and triglyceride and total cholesterol. The microarray data showed that expression differences among the 1131 genes, 636 genes were up-regulated and the other 495 genes are down regulated expression and immune / inflammatory genes in.675 In response, apoptosis, oxidative stress, metabolic process, cell cycle, cell proliferation, cytoskeleton, signal transduction, transcription, translation and transportation. Especially the expression level of Nt5e CeCl_3 significantly decreased after long-term exposure, causing liver inflammation reaction, and Cyp4a12a expression and low Cdkn1a expression leads to liver metabolic disorders.
(4) studies have demonstrated that Ln could induce apoptosis of mouse spleen cells and reduce its immunity, but its molecular mechanism is still uncertain. The molecular mechanism of apoptosis caused by exposure of spleen in mice for the study of Ln, this experiment at different doses (2,10 and 20mg/kg BW) treated with CeCl_3 60 days continuous irrigation, analysis of Ce accumulation. Spleen cell apoptosis and apoptosis related gene expression and protein from cells. Results showed that long-term exposure to CeCl_3 in the spleen of mouse Ce element significantly accumulated resulted in a significant increase in spleen coefficient and spleen cell apoptosis. Moreover, CeCl_3 increased the phosphorylation of Caspase-3 and caspase-9, inhibition of Bcl-2 gene and protein expression level of reactive oxygen species (ROS).. CeCl_3 is induced by splenic cell apoptosis via the intrinsic pathway.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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