四氧化三鐵是在生醫領域經常使用的金屬氧化物，由於其具有超順磁的特性，因此被廣泛應用在生醫領域，例如 MRI影像偵測，熱治療，藥物傳遞等。此材料已被廣泛應用於人體，因此，許多研究團隊對於四氧化三鐵是否會對細胞造成毒性均進行相關探討。然而， 不同團隊對於四氧化三鐵是否會對細胞造成毒性有不同的報導。四氧化三鐵間接對於細胞造成的傷害，是來自於當其解離後所生成的二價鐵離子與細胞內過氧化氫反應，所生成的氫氧自由基對細胞產生破壞所致。氫氧自由基是一種活性極高的活性氧，會破壞細胞內的膜狀胞器，引發細胞走向凋亡，而造成毒性。由Fe3O4 + 2H --> γFe2O3 + Fe2+ + H2O反應式中可知，四氧化三鐵解離程度與環境中pH值有關，因此，本實驗室推測四氧化三鐵經胞飲後所經過的不同胞器內部的pH值會影響四氧化三鐵的解離程度，進而影響氫氧自由基的生成能力。為了進一步釐清四氧化三鐵於各不同胞器環境中催化生成自由基的能力，我們利用具有熱敏感性之產氣微脂體做為包覆載體，使四氧化三鐵可以在定點釋放。我們將包覆有四氧化三鐵之微脂體餵食細胞，並於不同時間點加熱，以觀察四氧化三鐵於不同胞器環境下釋放對於其產生之氫氧自由基進而引發細胞死亡的影響。由本篇論文的實驗結果得知，四氧化三鐵在pH值較低（lysosome）的胞器環境中釋放，細胞的死亡率會因產生過多自由基而較顯著，反之，pH值較高的環境（endosome）死亡率則較不明顯。因此，未來將四氧化三鐵應用於生醫領域研究部分，可視其應用目的而選擇其於細胞內釋放位置，以達到最大效用。

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used for various biomedical applications, for example, magnetic resonance imaging, targeted delivery of drugs, and in hyperthermia. Because the materials is applied to human, the cytotoxicity of SPIONs is discussed by many research teams. However, they hold different opinions. SPIONs induce toxicity via the generation of hydroxyl radicals. SPIONs are dissociated iron ions with H+ ions, the free iron in the form of ferrous ions (Fe2+) can react with hydrogen peroxide produce highly reactive hydroxyl radicals via the Fenton reaction: Fe2++H2O2--> Fe3++OH_+OH. . We know the dissociation extent of SPIONs is related with pH value from Fe3O4 + 2H  γFe2O3 + Fe2+ + H2O, therefore, we consider the dissociation of SPIONs is different at various organelle and lead to production of intracellular hydroxyl radicals and cytotoxicity are different. In order to check the SPIONs ‘s catalysis production of hydroxyl radicals at different pH organelle. we use the thermal-sensitive liposome system as carrier to encapsulate SPIONs and control SPIONs to different organelle, after that, we observe the variation of cytotoxicity. Our results had shown that release SPIONs in lysosome leads to more hydroxyl radicals and significant cytotoxicity. It is encouraging to conclude the pH value in lysosome is benefit to SPIONs catalysis and directly induce cell death.

目錄
摘要
圖錄
表錄
第一章 緒論
1-1前言
1-2四氧化三鐵(Superparamagnetic iron oxide nanoparticles)
1-3 ROS(Reactive Oxygen Species)
1-4 DMT1 (divalent metal transporter 1)
1-5 Fenton Reaction
1-6過脂化(Lipid Peroxidation)
1-7細胞凋亡(Apotosis) and 細胞壞死(Necrosis)
1-8胞吞作用 (Endocytosis)
1-9 碳酸氫銨(Ammonium bicarbonate)
1-10 微脂體
第二章 實驗藥品與儀器
2-1 實驗藥品
2-2 實驗儀器
2-3本系統微脂體製備
2-3-1內部未包覆四氧化三鐵之微脂體製備(lip.)
2-3-2內部包覆四氧化三鐵之微脂體製備(mag-lip.)
2-4檢測螢光四氧化三鐵接枝度以及定量四氧化三鐵的包覆率
2-5測量四氧化三鐵在不同pH值解離成二價鐵離子的程度
2-6測量四氧化三鐵在不同pH值溶液中產生氫氧自由基的程度
2-7 HT-1080對Mag-lip.的吞噬情形
2-7-1 Mag-lip.隨著不同餵食時間在細胞的累積量
2-7-2載體於胞內傳輸路徑之探討
2-8 Mag-lip.對於細胞死亡率的影響
2-8-1定量分析(MTS Assay)
2-8-2定性分析 (Live&Dead)
2-9 Mag-lip.造成細胞死亡的方式
2-10 Mag-lip.在細胞體內產生二價鐵離子的數量
2-10-1定量分析(MTS Assay)
2-10-2 定性分析(Live&Dead)
2-11 Mag-lip.在細胞體產生活性氧(ROS)數量
2-11-1定量分析(Flow Cytometry)
2-11-2 定性分析(CLSM)
2-12 Mag-lip.在細胞體內產生氫氧自由基的數量
2-13 Mag-lip.在細胞體所造成過氧化的程度
2-13-1定量分析
2-13-2定性分析
第三章 實驗結果與討論
3-1四氧化三鐵特性探討
3-1-1四氧化三鐵粒徑以及電荷大小
3-1-2四氧化三鐵在不同pH值解離能力
3-1-3四氧化三鐵在不同pH值產生氫氧自由基能力
3-1-4 選擇最佳化四氧化三鐵濃度
3-1-5四氧化三鐵螢光接枝
3-2微脂體特性探討
3-2-1 微脂體粒徑以及電荷大小
3-2-2 微脂體內四氧化三鐵包覆率
3-2-3 HT1080對Mag-lip.的吞噬能力
3-2-4 HT1080細胞吞噬Mag- liposome之路徑探討
3-3環境溫度及材料本身毒性對細胞存活率之影響
3-4四氧化三鐵在不同胞器環境釋放對於細胞死亡率影響
3-5四氧化三鐵所催化氫氧自由基造成細胞死亡方式之探討
3-6四氧化三鐵於不同胞器內解離能力
3-7四氧化三鐵於不同胞器內誘發ROS之分析
3-8四氧化三鐵於不同胞器內誘發氫氧自由基之分析
3-9四氧化三鐵於不同胞器內釋放造成細胞內過氧化程度的差異
3-9.1 定量分析細胞內過氧化差異
3-9.2 定性分析細胞內過氧化差異
第四章 結論
參考資料

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