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研究生: 郭禹萱
Yu-Xuan Guo
論文名稱: 肝素化瓊脂糖微球之製備及其應用於親和純化外泌體之研究
Development of heparinzed agarose microspheres for affinity purification of exosomes
指導教授: 蔡協致
Hsieh-Chih Tsai
口試委員: 高震宇
許銘賢
林宣因
學位類別: 碩士
Master
系所名稱: 應用科技學院 - 應用科技研究所
Graduate Institute of Applied Science and Technology
論文出版年: 2023
畢業學年度: 112
語文別: 中文
論文頁數: 96
中文關鍵詞: 細胞外囊泡外泌體微球瓊脂糖肝素親和性純化
外文關鍵詞: extracellular vesicle, exosome, microsphere, agarose, heparin, affinity purification
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  • 致謝 I 摘要 II ABSTRACT III 目錄 V 圖目錄 IX 表目錄 XII 第一章 前言 1 第二章 文獻回顧 2 2.1 細胞外囊泡 2 2.1.1 何謂細胞外囊泡 2 2.1.2 外泌體的應用 3 2.1.2.1 藥物輸送系統 3 2.1.2.2 組織再生 4 2.1.2.3 疾病診斷 5 2.1.2.4 癌症免疫療法 6 2.1.3 外泌體分離純化方法 8 2.1.3.1 差速離心法 8 2.1.3.2 高分子共沉澱法 10 2.1.3.3 薄膜過濾法 11 2.1.3.4 尺寸排除層析法 12 2.1.3.5 免疫親和法 13 2.1.4 外泌體檢測方法 14 2.1.4.1 形貌表徵 14 2.1.4.2 尺寸分佈和顆粒濃度 15 2.1.4.3 蛋白質檢測 16 2.1.4.4 外泌體特定標記檢測 17 2.3 微球 20 2.3.1 微球定義 20 2.3.2 微球製備方法 20 2.3.3 乳化反應 23 2.3.4 乳化劑介紹 24 2.3.5 親水親油平衡值 25 2.4 水凝膠 26 2.4.1 瓊脂糖簡介 26 2.4.2 瓊脂糖交聯方法 27 2.5 肝素 28 2.5.1 肝素簡介 28 2.5.2 肝素應用 30 第三章 實驗方法 32 3.1 實驗流程 32 3.2 實驗藥品 33 3.3 實驗設備 35 3.4 實驗儀器 36 3.5 實驗步驟 37 3.5.1 乳化法製備瓊脂糖微球 37 3.5.2 瓊脂糖微球表面改質環氧基團 38 3.5.3 環氧化瓊脂糖微球鍵結肝素 39 3.6 結構鑑定與性質分析 39 3.6.1 微球粒徑量測 39 3.6.2 微球膨潤度及含水率測試 40 3.6.3 環氧當量測試 40 3.6.4 肝素含量測試 41 3.7 細胞實驗 42 3.7.1 細胞培養 42 3.7.2 細胞存活率測試 42 3.7.3 外泌體分離 44 3.7.3.1 尺寸排除層析法 45 3.7.3.2 親和捕捉法 45 3.7.4 外泌體定性定量分析 46 3.7.4.1 粒徑分佈及顆粒濃度 46 3.7.4.2 蛋白質含量測定 47 3.7.4.3 表面型態鑑定 48 3.7.4.4 免疫特徵鑑定 48 3.7.4.5 外泌體標記和追蹤 49 第四章 結果與討論 51 4.1 具肝素之親和微球之研究 51 4.1.1 乳化法製備瓊脂糖微球之研究 51 4.1.1.1 粒徑分析 51 4.1.1.2 表面形貌分析 54 4.1.2 瓊脂糖微球表面改質環氧基團之研究 55 4.1.2.1 衰減全反射式傅立葉紅外(ATR-FTIR)光譜分析 56 4.1.2.2 拉曼光譜(Raman spectroscopy)分析 57 4.1.2.3 能量散射X射線光譜(Energy Dispersive X-ray Spectrometer)分析 58 4.1.2.4 瓊脂糖微球表面環氧基團含量分析 61 4.1.2.5 粒徑分析 62 4.1.3 環氧化瓊脂糖微球鍵結肝素之研究 63 4.1.3.1 衰減全反射式傅立葉紅外(ATR-FTIR)光譜分析 64 4.1.3.2 拉曼光譜(Raman spectroscopy)分析 65 4.1.3.3 能量散射X射線光譜(Energy Dispersive X-ray Spectrometer)分析 66 4.1.3.4 環氧化瓊脂糖微球鍵結肝素含量分析 69 4.1.3.5 粒徑分析 72 4.1.3.6 表面形貌分析 74 4.1.3.7 膨潤度及含水率測試 75 4.2 生物相容性試驗 77 4.2.1 微球對於細胞純活率之研究 77 4.3 細胞培養液外泌體純化 78 4.3.1 外泌體粒徑大小分佈及濃度 78 4.3.2 外泌體純度及回收率 80 4.3.3 外泌體表面形貌 81 4.3.4 外泌體免疫特徵 82 4.3.5 外泌體標記和追蹤 83 第五章 結論 86 第六章 參考文獻 88

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