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研究生： 林天宇
Tian-Yu Lin
論文名稱： 錳離子在ICR小鼠視網膜毒性之研究
Toxicity of Manganese ions to ICR mice retina
指導教授： 吳忠信
Wu, Chung-Hsin
學位類別： 碩士
Master
系所名稱： 生命科學系
Department of Life Science
論文出版年： 2014
畢業學年度： 102
語文別： 中文
論文頁數： 103
中文關鍵詞： 錳 、視網膜 、眼瞼下注射 、氯化錳 、ICR小鼠 、視網膜電位 (ERG) 、細胞凋亡(apoptosis)
英文關鍵詞： manganese, retina, subconjunctiva injected, MnCl2, ICR mice, electroretinogram (ERG), apoptosis
論文種類： 學術論文
相關次數： 點閱：82  下載：4 
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    • 本研究主要在於探討過量錳離子對ICR小鼠視網膜功能之影響。錳離子在動物體內需求極微量但卻不可或缺，過量的錳進入體內會造成中毒。過去對錳中毒的研究大多著重在呼吸系統、中樞神經系統損傷及發炎等臨床現象，對於視覺系統的研究非常少，對於錳的急性影響研究更少。本研究是利用眼瞼下注射 (subconjunctival injection)的方式，將生理食鹽水（control)、40 mM MnCl2 及 80 mM MnCl2注入眼球，觀察注射後在第一天、第三天及第七天，以不同強度光源的刺激，觀察小鼠視網膜電位 (ERG) a-wave與b-wave的振幅與隱含期的反應，以評估檢測視網膜功能是否受到影響。進一步再利用H&E染色，評估視網膜各層構造，探討視網膜的哪一層細胞可能受到影響，也利用免疫組織染色技術，以cleaved caspase-9、cleaved caspase-3、cleaved PARP、TNF-α、Bax、Bcl-2等抗體，來評估視網膜細胞受傷害的程度，結果顯示，眼瞼下注射不論在40 mM或80 mM MnCl2的濃度，確實都會造成視網膜功能受損，結果顯示ERG的a-wave 和 b-wave振幅 (amplitude)降低、隱含期 (implicit tim)延長，視網膜組織受損，從ChAT表現推測眼壓應沒有升高，免疫染色呈現視網膜可能發炎，也不能排除細胞發生凋亡的可能性。這些結果顯示MnCl2處理眼球，短時間的急性毒害可能是引起視網膜發炎、甚至細胞凋亡，因而引起ERG的a-wave與b-wave發生改變。

    The purpose of the present study was to assess the toxic effect of manganese (Mn) to the retina of ICR mice. Manganese is essential for human and animal bodies although its requirement is very minor. Ex-cess accumulation of Mn in the animal body may induce toxic to a vari-ety of tissues. The study of Mn toxic effect are most concentrated in the respiratory system and central nervous system in the clinic as well as the inflammatory effect. The toxic effect of Mn on the visual system is still to be uncertain. To examine the toxic effect of Mn on the retina, MnCl2 was microinjected into the subconjunctiva of both sides in the ICR mice. The animal was received two different concentrations of MnCl2 solution, 40 mM and 80 mM, and saline as the control. On the first, third and seventh days after injection, the electroretinogram (ERG) of all experimental mice was examined in response to the low and high illumination to evaluate the function of the retina. After ERG examina-tion, the retina of both eyes of each mouse were dissected and fixed, were then stained with hematoxylin and eosin (H&E) and immunofluo-rescence with the antibodies of Cleaved Caspase-9,Cleaved Caspase-3,Cleaved PARP,TNF-α, Bax, Bcl-2 and ChAT. We found that MnCl2 treatment produced a decrease in the amplitude and a simultaneous delay of the implicit time of both the a-wave and b-wave caused by the low and high intensity of luminous. Histological data demonstrated that the retinal cells, specially in the inner nuclear layer and ganglionic layer, probably loss. Data from the immunochemical studies had shown that the TNF- and apoptotic proteins were expressed in the retina. These data strongly suggest that malfunction of the retina caused by MnCl2 treatment may be due to the pathological changes of the neurons in the retina because of their inflammation and apoptosis.

    中文摘要••••••••••••••••••••••••••••••••••••••••••••••••••••5 英文摘要••••••••••••••••••••••••••••••••••••••••••••••••••••6 一、前言••••••••••••••••••••••••••••••••••••••••••••••••••••8 （一）錳的來源和分布••••••••••••••••••••••••••••••••••••••••••8 （二）錳在體內的的生理作••••••••••••••••••••••••••••••••••••••8 （三）高濃度錳汙染的致病路••••••••••••••••••••••••••••••••••••9 （四）高濃度錳汙染致病機•••••••••••••••••••••••••••••••••••••10 （五）細胞凋亡(apoptosis)•••••••••••••••••••••••••••••••••••11 （六）高濃度錳對眼睛的損害•••••••••••••••••••••••••••••••••••12 （七）視網膜電位圖 (Electroretinogram，ERG)••••••••••••••••••13 （八）研究的問題••••••••••••••••••••••••••••••••••••••••••••14 二、研究的材料與方法•••••••••••••••••••••••••••••••••••••••••16 （一）實驗動物••••••••••••••••••••••••••••••••••••••••••••••16 （二）氯化錳 (MnCl2) 注射•••••••••••••••••••••••••••••••••••16 （三）視網膜電圖記錄 (ERG recording)•••••••••••••••••••••••••17 （四）病理切片••••••••••••••••••••••••••••••••••••••••••••••18 1. H&E染色法步驟•••••••••••••••••••••••••••••••••••••19 2. 免疫螢光法步驟•••••••••••••••••••••••••••••••••••••20 （五）藥物的配製••••••••••••••••••••••••••••••••••••••••••••21 （六）資料的分析與統計•••••••••••••••••••••••••••••••••••••••23 三、結果•••••••••••••••••••••••••••••••••••••••••••••••••••25 （一） MnCl2對於ICR小鼠ERG之影響•••••••••••••••••••••••••••••25 1. 比較不同濃度MnCl2於注射後第一天、第三天、第七天對a-wave的影響••••••••••••••••••••••••••••••••••••••••••••••••••••••26 2. 比較不同濃度MnCl2在低強度與高強度光源刺激下，ERG中a-wave的反應••••••••••••••••••••••••••••••••••••••••••••••••••••27 3. 比較不同濃度MnCl2於注射後第一天、第三天、第七天，對b-wave的影響••••••••••••••••••••••••••••••••••••••••••••••••••••29 4. 比較不同濃度MnCl2在低強度與高強度光源刺激下，ERG中b-wave的反應••••••••••••••••••••••••••••••••••••••••••••••••••••31 （二） H&E染色及ChAT與TNF-α免疫螢光染色結果•••••••••••••••••••33 1. H&E染色••••••••••••••••••••••••••••••••••••••••••33 2. ChAT 免疫螢光染•••••••••••••••••••••••••••••••••••34 3. TNF-α免疫螢光染•••••••••••••••••••••••••••••••••••35 （三） Apoptosis細胞凋亡因子免疫螢光染色結果••••••••••••••••••36 1. Cleaved Caspase-9 免疫螢光染色••••••••••••••••••••37 2. Cleaved Caspase-3免疫螢光染色•••••••••••••••••••••38 3. Cleaved PARP 免疫螢光染色•••••••••••••••••••••••••38 4. Bax免疫螢光染色•••••••••••••••••••••••••••••••••••39 5. Bcl-2免疫螢光染色•••••••••••••••••••••••••••••••••40 四、討論•••••••••••••••••••••••••••••••••••••••••••••••••••41 （一） 將MnCl2 以subconjunctival injection方式，造成視網膜的ERG與組織構造發生改變是一項實驗技術••••••••••••••••••••••••••••••41 （二） MnCl2可能藉由損傷感光細胞而引起視網膜a-wave高度降低••••••42 （三） MnCl2可能藉由損傷視網膜ONL層上的細胞而引起視網膜b-wave高度降低••••••••••••••••••••••••••••••••••••••••••••••••••••••••46 （四） MnCl2可能僅在注射初期才會引起視網膜電位的implicit time延後 ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••47 五、結論•••••••••••••••••••••••••••••••••••••••••••••••••••49 六、參考文獻•••••••••••••••••••••••••••••••••••••••••••••••50 七、圖表與圖說••••••••••••••••••••••••••••••••••••••••••••••58

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