臺灣博碩士論文加值系統

紅血球細胞膜由脂質和蛋白質組成，具有生物相容性和生物識別性，被廣泛研究作為貨物載體。紅血球細胞膜能夠通過表面的醣蛋白質特異性地與標的細胞相互作用，實現藥物的靶向傳遞，並且可以規避免疫系統的吞噬，提高穩定性和療效。
以往製備紅血球細胞膜囊泡的方法比較複雜，如表面結合、封裝和機械擠壓等。本研究簡化了製備過程，使用超音波技術製備細胞膜囊泡，並將螢光染劑封裝進紅血球細胞膜內。這種方法不僅簡單高效，而且能夠確保染劑的有效包覆和囊泡的一致性。
我們使用可見光激發（400 nm-760 nm）的LED與雷射光源來觀察包裹染劑的紅血球細胞膜囊泡的螢光放光成像。光源的選擇對亮度排序影響很大，主要是由於光源特性和樣品對不同波長光的吸收和發射特性不同。我們比較了不同染劑的放光強度、在細胞膜的穩定性以及對細胞型態的影響。
此外，人類紅細胞表面含有不同血型的抗原，例如A型血表面有A型抗原。實驗中，將封裝螢光染劑的A型紅血球細胞膜與A抗體結合，觀察到凝集現象，證明封裝過染劑的細胞膜表面A型抗原仍存在。這為紅血球表面抗原的檢測提供了一種新的逆向檢測方法。

The red blood cell membrane is composed of lipids and proteins, possessing biocom-patibility and bio-recognition properties. This makes it widely studied as a cargo car-rier. The red blood cell membrane can specifically interact with target cells through glycoproteins on its surface, achieving targeted drug delivery. Additionally, it can evade phagocytosis by immune cells such as macrophages, further enhancing the sta-bility and efficacy of the cargo carrier.
Previously, the preparation methods for red blood cell membrane vesicles were rela-tively complex, involving surface binding, encapsulation, and mechanical extrusion. This study simplifies the preparation process by using ultrasonic technology to pro-duce cell membrane vesicles and encapsulate fluorescent dyes within the red blood cell membranes. This method is not only simple and efficient but also ensures effec-tive dye encapsulation and vesicle consistency.
We used LED and laser light sources with visible wavelengths (400 nm-760 nm) to observe the fluorescence imaging of the dye-encapsulated red blood cell membrane vesicles under a microscope. The choice of light source significantly affects the brightness ranking, primarily due to the characteristics of the light source and the ab-sorption and emission properties of the sample at different wavelengths. We compared the emission intensity of different dyes, their stability within the cell membrane, and their impact on cell morphology.
Moreover, human red blood cells have surface antigens that can be classified into dif-ferent blood types, such as A-type blood cells having A-type antigens on their surface. In our experiment, we combined fluorescent dye-encapsulated A-type red blood cell membranes with A antibodies and observed agglutination. This demonstrates that the A-type antigens on the cell membrane surface remain intact after dye encapsulation. This provides a new reverse detection method for detecting antigens on the surface of red blood cells.

論文審定書 i
謝誌 ii
摘要 iii
Abstract iv
目錄 vi
圖目錄 ix
表目錄 xiv
縮寫用語對照表 xv
1. 前言 1
1.1紅血球細胞膜表面蛋白質回顧 1
1.2血型簡介 2
1.3血型測定 5
1.3.1 引發紅血球凝集的基本機轉 8
1.3.2 鈎狀效應 8
1.4紅血球細胞膜囊泡的製備與應用 10
1.5紅血球運輸載體回顧 11
1.6紅血球細胞膜自組裝作用 13
1.7空化效應（Cavitation Effect） 14
1.8常見的螢光染劑 15
1.9本研究使用的螢光染劑 16
1.10凝膠過濾法（Gel Filtration） 20
1.10.1 G-25 脫鹽純化管柱（G-25 Desalting Spin Columns） 20
1.11抗體檢測方法 21
1.12逆向檢測（Reverse Testing） 22
1.13研究動機 23
2. 實驗部分 24
2.1實驗藥品 24
2.2儀器步驟 26
2.2.1 倒立式螢光顯微鏡（Inverted Fluorescence Microscopy） 26
2.2.1.1 以LED為激發光源 26
2.2.1.2 以雷射光為激發光源 30
2.2.2 紫外-可見光譜儀（Ultraviolet-visible Spectroscopy, UV-Vis） 33
2.2.3 離心機（Centrifuge） 33
2.2.4 單眼相機 33
2.2.5 迷你超音波破碎儀 33
2.2.6 掃描式電子顯微鏡Scanning Electron Microscope，SEM） 33
2.3溶液配置 34
2.3.1 磷酸緩衝溶液（Phosphate Buffered Saline） 34
2.3.2 螢光染劑溶液 34
2.3.3 血球抗體溶液 35
2.3.3.1 凝膠過濾法（Gel Filtration） 36
2.3.3.2 G-25 脫鹽純化管柱（G-25 Desalting Spin Columns） 36
2.4樣品製備 37
2.5製備方法最佳化 39
2.5.1 最佳化去除血紅蛋白的緩衝溶液濃度 39
2.5.2 最佳化去除血紅蛋白次數 46
2.6玻片前處理 52
2.6.1 清洗蓋玻片與載玻片 52
2.6.2 以APTES 處理載玻片 52
2.7迷你超音波破碎儀樣品製備 53
2.8倒立式螢光顯微鏡樣品製備 53
2.9掃描式電子顯微鏡樣品製備 53
3. 結果與討論 54
3.1紅細胞膜囊泡包覆螢光染劑的放光成像比較 54
3.2紅細胞膜囊泡包覆染料後的穩定性測試 60
3.3細胞實驗與抗原測定 66
3.4 以雷射光作為激發光源觀察囊泡的放光成像 69
3.5 紅細胞膜囊泡包覆螢光染劑之掃描式電子顯微鏡成像 81
4. 結論 88
5. 未來展望 89
6. 參考文獻 90

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