紅血球 (Red Blood Cell, Erythrocyte) 是人體中極為重要的細胞，主要負責攜帶氧氣至全身各處細胞並代謝二氧化碳。而奈米粒子 (特別是銀奈米粒子) 則因為具有特殊的物理及化學特性，如今廣泛應用於抗菌產品、細胞標定及藥物載體等生醫用途，因此釐清銀奈米粒子對紅血球細胞的外觀、細胞骨架結構與機械性質的影響是十分重要的課題。本研究利用銀奈米粒子對大鼠紅血球細胞作用，輔以光學顯微鏡、原子力顯微鏡與穿透式電子顯微鏡觀察紅血球外觀的改變情形；並利用原子力顯微鏡及電子顯微鏡觀察紅血球細胞骨架結構變化情形；最後以原子力顯微鏡量測紅血球彈性模數的變化，釐清銀奈米粒子對紅血球骨架與血球機械性質的影響。
研究發現，銀奈米粒子作用後的紅血球，外觀由正常的雙凹圓盤狀變成棘刺狀並且伴隨溶血現象發生；細胞骨架則會從正常的均勻分佈，轉為聚集且骨架直徑增加。由原子力顯微鏡量測紅血球的機械性質，發現在銀奈米粒子作用後，紅血球的彈性模數明顯高於正常的紅血球。由實驗結果得知，紅血球受到銀奈米粒子作用後，其細胞骨架將產生聚集且變粗，導致血球的彈性模數上升，進而影響血球的變形能力，於使用時須加以注意。

Erythrocytes (red blood cells, RBCs) are important cells in human bodies, and they are responsible for transporting oxygen and metabolizing carbon dioxide. Besides, nanoparticles (NPs) (especially Ag NPs) are widely applied to biomedical fields such as anti-bacteria products, cell labeling, and drug carriers because of their special physical and chemical properties. It is an important issue to clarify how Ag NPs affects the morphology, cytoskeleton structure and mechanical property of RBCs. Hence in this study, the morphology of RBCs under the influence of Ag NPs was observed using an optical microscope (OM), an atomic force microscope (AFM), and a transmission electron microscope (TEM). The cytoskeleton structure was observed by AFM and TEM. The mechanical behavior was characterized by Bio-AFM. The correlation between the change in cytoskeletal and the difference in mechanical behavior was examined. Experimental results indicated that Ag-NP-affected RBCs became echinocyte-like rather than biconcave and were accompanied with hemolysis. The crosslinking density and diameter of cytoskeleton increased and the cytoskeleton structure became more agglomerated. The AFM indentation indicated that Ag-NP-affected RBCs had an increased elastic modulus. In summary, under the influence of Ag NPs, the stiffness of RBCs increases and the deformability decreases in consequence of cytoskeleton aggregation and thickening that is concerned with the use of Ag NPs.

摘要..............................................................I
Abstract........................................................II
誌謝............................................................III
壹、前言..........................................................1
貳、文獻回顧......................................................2
2-1 紅血球與細胞骨架...........................................2
2-1-1 紅血球 (Erythrocytes).....................................2
2-1-2 細胞骨架 (Cytoskeleton)...................................6
2-1-3 細胞骨架聚合解聚...........................................9
2-1-4 細胞骨架與細胞受力變形....................................14
2-1-5 影響紅血球的因素.........................................19
2-2 奈米粒子.................................................25
2-2-1 奈米粒子對細胞作用........................................29
2-2-2 奈米粒子對細胞骨架的影響...................................33
2-2-3 銀奈米粒子對紅血球的細胞毒性...............................38
2-2-4 銀奈米粒子對紅血球外觀的影響...............................41
2-2-5 銀奈米粒子對紅血球細胞骨架的影響...........................44
2-2-6 銀奈米粒子對紅血球機械性質的影響...........................47
2-3 紅血球與細胞骨架形貌觀察...................................49
2-3-1 原子力顯微鏡 (AFM).......................................53
2-3-2 穿透式電子顯微鏡 (TEM)....................................58
2-4 細胞機械行為分析..........................................64
2-5 研究目的.................................................75
參、實驗步驟.....................................................76
3-1 實驗流程.................................................76
3-2 紅血球溶液製備............................................78
3-2-1 磷酸鹽緩衝溶液............................................78
3-2-2 紅血球溶液製備............................................78
3-2-3 甲醛固定液...............................................79
3-2-4 四氧化鋨固定液............................................79
3-3 紅血球試片製備............................................79
3-3-1 紅血球形貌觀察............................................79
3-3-2 紅血球組織固定、脫水......................................80
3-3-3 原子力顯微鏡試片..........................................81
3-3-4 穿透式電子顯微鏡試片......................................82
3-4 分析儀器.................................................83
3-4-1 倒立式光學顯微鏡 (OM).....................................83
3-4-2 氣相原子力顯微鏡 (AFM in air).............................83
3-4-3 液相原子力顯微鏡 (AFM in fluid)...........................84
3-4-4 生物型穿透式電子顯微鏡 (Bio-TEM)..........................84
3-4-5 場發射穿透式電子顯微鏡 (FE-TEM)...........................85
3-4-6 微量離心機 (Microcentrifuge).............................85
3-5 原子力顯微鏡實驗參數......................................85
3-5-1 原子力顯微鏡氣相掃描......................................85
3-5-2 原子力顯微鏡液相掃描......................................86
3-5-3 原子力顯微鏡機械性質量測...................................86
肆、結果與討論....................................................88
4-1 紅血球形貌觀察............................................88
4-1-1 OM觀察紅血球形貌..........................................88
4-1-2 AFM氣相觀察紅血球形貌.....................................90
4-1-3 AFM液相觀察紅血球形貌.....................................97
4-1-4 TEM液相觀察紅血球形貌....................................104
4-2 紅血球細胞骨架觀察.......................................107
4-2-1 AFM氣相觀察紅血球細胞骨架................................107
4-2-2 AFM液相觀察紅血球細胞骨架................................121
4-2-3 TEM液相觀察紅血球細胞骨架................................134
4-3 紅血球機械性質量測.......................................138
4-3-1 AFM液相量測紅血球機械性質................................138
4-3-2 AFM、Nanoindenter、微吸管量測紅血球機械性質比較...........146
伍、結論........................................................150
陸、參考文獻....................................................152

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