本文關鍵詞： 稀土 肝損傷 炎癥 免疫 細胞凋亡 氧化應激 基因表達譜　出處：《蘇州大學》2013年碩士論文　論文類型：學位論文

【摘要】：隨著稀土應用的日趨廣泛，不可避免地進入環(huán)境中并在生物體內(nèi)累積，其對人體健康可能造成的影響己引起人們的普遍關注。作為輕稀土元素最重要的蓄積器官，稀土對肝臟的損傷作用值得深入探討。目前，國內(nèi)外已開展稀土元素對肝毒性和免疫功能影響的研究，但其分子機制尚未清楚。鑒于此，本論文主要研究稀土長期攝入對肝臟及機體免疫功能的影響，為制定稀土使用安全標準和評價稀土生物效應提供實驗依據(jù)。 論文主要涉及以下內(nèi)容： （1）鑭系元素（Ln）可以對大鼠和小鼠產(chǎn)生各種毒性作用，但是其作用機制仍不明朗。本實驗中，用等離子電感耦合-質(zhì)譜、不同光譜方法、凝膠電泳及透射電子顯微鏡的技術手段，以不同劑量（2、10和20mg/kg BW）的CeCl_3持續(xù)灌胃小鼠45天觀察肝臟DNA構象變化及肝細胞凋亡程度。結(jié)果表明CeCl_3處理后小鼠肝體比顯著上升。肝臟中鈰元素明顯累積，并插入到DNA堿基對中或與DNA核苷酸結(jié)合，從而改變DNA構象。此外，通過凝膠電泳及透射電子顯微鏡觀察，高劑量的Ce~(3+)可導致DNA斷裂和肝細胞凋亡。 （2）已有的研究證實Ln可引起小鼠肝損傷，然而其引發(fā)凋亡的分子機制仍不清楚。本實驗以不同劑量（2、10和20mg/kg BW）的CeCl_3持續(xù)灌胃小鼠60天，分析肝細胞超微結(jié)構變化、氧化脅迫水平、多種氧化應激物、酶以及與應激有關的基因表達水平的變化情況。研究發(fā)現(xiàn)，CeCl_3暴露60天后，肝臟中鈰元素明顯累積，從而引發(fā)肝細胞凋亡。CeCl_3顯著促進活性氧（ROS）的產(chǎn)生，并抑制應激相關基因（超氧化物歧化酶、過氧化氫酶、谷胱甘肽過氧化物酶、金屬硫蛋白、熱休克蛋白70、谷胱甘肽-硫-轉(zhuǎn)移酶、P53和鐵轉(zhuǎn)運蛋白）的表達水平；同時有效地激活細胞色素p4501A的表達。這些結(jié)果表明CeCl_3可以導致小鼠肝細胞凋亡，并改變與金屬排毒/代謝調(diào)節(jié)和自由基清除有關基因的表達水平。 （3）Ln在肝中累積，并造成一系列損傷，但對Ln造成的肝損傷與基因表達譜的關系研究卻知之甚少。以2mg/kg BW劑量CeCl_3連續(xù)灌胃處理雄性小鼠90天，結(jié)合基因芯片技術，分析肝損傷及基因表達的變化情況。結(jié)果發(fā)現(xiàn)長期暴露后導致Ce元素在肝中累積，引發(fā)肝炎癥反應和肝細胞壞死。同時，Ce累積使白細胞、淋巴細胞、血小板、網(wǎng)織紅細胞（Ret）和嗜中性粒細胞及A/G比率明顯下降；而堿性磷酸酶、乳酸脫氫酶和膽堿酯酶活性及甘油三酯和總膽固醇的濃度顯著上升。基因芯片數(shù)據(jù)顯示有1131個基因差異表達，其中，636個基因顯著性上調(diào)，，其余495個基因顯著性下調(diào)。675個已知功能基因的表達變化與免疫/炎癥反應、細胞凋亡、氧化應激、代謝過程、細胞周期、細胞增殖、細胞骨架、信號傳導、轉(zhuǎn)錄、翻譯和運輸有關。尤其CeCl_3長期暴露后Nt5e的表達水平顯著下調(diào)，引發(fā)肝炎癥反應，而Cyp4a12a的過表達和Cdkn1a的低表達導致肝代謝紊亂。 （4）已有研究證明Ln可以導致小鼠脾細胞凋亡，并降低其免疫能，但其分子機制仍不明朗。為研究Ln暴露造成小鼠脾凋亡的分子機制，本實驗以不同劑量的（2、10和20mg/kg BW）CeCl_3連續(xù)灌胃處理60天，分析Ce累積、脾細胞凋亡及凋亡相關細胞因基因和蛋白的表達情況。結(jié)果發(fā)現(xiàn)長期CeCl_3暴露后小鼠脾臟中Ce元素明顯累積，導致脾系數(shù)顯著上升和脾細胞凋亡。而且，CeCl_3顯著激活caspase-3和caspase-9，抑制Bcl-2的基因和蛋白表達水平，促進活性氧（ROS）的產(chǎn)生。說明CeCl_3是通過內(nèi)在途徑誘導脾細胞凋亡。
[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.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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