紅血球，作為人體內重要的工作細胞，攜帶氧氣至各器官組織並攜出二氧化碳以利於代謝循環，具有極佳的變形能力。銀奈米粒子因具有顯著的抗菌能力，被廣為用於藥物載體、抗菌產品等。然而，過去研究顯示，銀奈米粒子進入人體，會引起細胞毒性，造成紅血球形貌改變，誘發細胞凋亡。但是，關於銀奈米粒子如何改變細胞骨架構造及作用後機械性質變化趨勢仍未被完整闡述，除此之外，銀奈米粒子作用後是否影響到紅血球的變形行為及其骨架結構皆值得被釐清。
本實驗以銀奈米粒子與紅血球作用，搭配原子力顯微鏡於液相環境下進行影像掃描，觀察作用後細胞形貌及細胞骨架結構變化；並搭配原子力顯微鏡液相力學檢測以行活體細胞彈性模數量測，了解機械性質的變化趨勢。除了研究未變形條件下，銀奈米粒子作用所帶來的影響外，本實驗亦還原紅血球於人體中循環的變形環境，使紅血球被動變形於微流道中，再行細胞骨架結構及活體細胞機械性質檢測。
研究結果發現，在未變形條件下，銀奈米粒子作用後細胞骨架變粗、密度增加且彈性模數上升；在變形條件下，作用後的細胞骨架出現片狀結構且骨架具有方向性，彈性模數上升趨勢大於正常紅血球。

Erythrocytes (red blood cells, RBCs) play an essential role in delivering oxygen from lung to the body tissues and facilitating the scrapping of carbon dioxide via metabolism. While navigating through microcirculation vessels and during spleen filtration, erythrocytes display excellent mechanical characteristics. Moreover, different from the bulk materials, nanomaterials have remarkable chemical properties. Among several kinds of nanoparticle (NPs), silver nanoparticles (Ag-NPs) have conspicuous properties of antibacterial, so widely used in drug delivery carriers and antibacterial. However, previous research found that not only nanoparticle is easier to be up taken by cells, but Ag-NPs pose for human health and lead to cytotoxicity, deformability of erythrocytes’ morphology, and then apoptosis. Therefore, it is a vital issue to clarify the interaction between erythrocytes and silver nanoparticles. Further research is requisite on the variation of mechanical properties and the structure of cytoskeleton.
In this study, exposure erythrocytes of SD rat to 200 μg/ml silver nanoparticles and phosphate buffered solution (PBS) respectively for 24 hours. After that we use atomic force microscopy (AFM) imaging to analyze the morphology and structure of erythrocytes’ cytoskeleton, and quantitative mechanical indentation to correlate structure and mechanical properties of non-fixed RBCs. Moreover, we manufacture microchannel devices simulating the capillary, the narrowest vein in the circulation, in order to investigate the influence on the deformed behavior of non-fixed cytoskeleton.
This experimental data reveals that the cytoskeleton interacting with Ag-NPs will become wider than with PBS, and the Young’s Modulus will raise with the density of cytoskeleton increasing. While the erythrocytes reacting to Ag-NPs deform in the microchannel, the structure of cytoskeleton becomes flake-shape, and the mechanical properties increase. This result demonstrates that silver nanoparticle will induce oxidation, which not alters the deformability of cytoskeleton, but also destroys the mechanical properties of erythrocytes.

致謝 I
摘要 III
Abstract IV
圖目錄 IX
表目錄 XXXI
壹、前言 1
貳、文獻回顧 3
2-1 紅血球 3
2-1-1 細胞結構 3
2-1-2 細胞骨架 4
2-1-3 影響紅血球細胞骨架的因素 5
2-2 奈米粒子對細胞的影響 16
2-2-1 銀奈米粒子 16
2-2-2 細胞骨架 17
2-2-3 紅血球細胞毒性 18
2-2-4 紅血球形貌與細胞骨架 19
2-2-5 紅血球機械行為 21
2-3 細胞骨架結構分析 41
2-3-1 原子力顯微鏡 44
2-3-2 原子力顯微鏡於生物應用 46
2-4 細胞機械性質 60
2-4-1 原子力顯微鏡 62
2-4-2 原子力顯微鏡於生物應用 64
2-5 研究目的 74
參、實驗步驟 75
3-1 實驗流程 75
3-2 紅血球溶液 77
3-2-1 磷酸鹽緩衝溶液 77
3-2-2 戊二醛溶液 77
3-2-3 紅血球溶液製備 78
3-3 紅血球試片製備 79
3-3-1 紅血球溶液固定處理 79
3-3-2 過氧化物檢測試片處理 79
3-3-3 OM影像觀察試片處理 81
3-3-4 TEM影像觀察試片處理 81
3-3-5 AFM影像觀察試片(未變形)處理 82
3-3-6 AFM壓痕力學分析試片(未變形)處理 82
3-3-7 AFM影像觀察試片(變形後)處理 82
3-3-8 AFM壓痕力學分析試片(變形後)處理 83
3-4 微流道製備 85
3-4-1 光罩設計 85
3-4-2 黃光微影製程 86
3-4-3 反應離子蝕刻製程 86
3-4-4 PDMS翻模 86
3-4-5 鍍覆蛋白膠 86
3-5 紅血球形貌及細胞骨架結構分析 88
3-5-1 穿透式電子顯微鏡 88
3-5-2 原子力顯微鏡 88
3-5-3 結構分析方法 89
3-6 紅血球機械性質分析 93
3-6-1 原子力顯微鏡 93
3-6-2 機械性質分析方法 93
肆、結果與討論 95
4-1 形貌觀察 95
4-1-1 紅血球形貌OM觀察 95
4-1-2 紅血球形貌AFM觀察 97
4-2 細胞骨架結構 106
4-2-1 細胞骨架結構AFM觀察 106
4-2-2 細胞骨架結構綜合分析 112
4-2-3 細胞骨架方向性綜合分析 126
4-3 細胞毒性檢測 133
4-3-1 過氧化物含量檢測 133
4-4 機械性質 135
4-4-1 紅血球之彈性模數 135
4-4-2 彈性模數與骨架結構綜合分析 144
4-5 穿透式顯微鏡 151
4-5-1 紅血球形貌TEM觀察 151
4-5-2 細胞骨架結構TEM觀察 154
伍、結論 159
陸、參考文獻 161
柒、附錄 171

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