胞外體(extracellular vesicles, EVs)是由細胞分泌到細胞外，能夠被受體細胞攝取的膜性囊泡小體。周圍由雙層磷脂質所包覆，直徑範圍可從30 nm到5,000 nm，且隨著分泌細胞的狀態，其分子組成也都不同。近年來越來越多研究指出胞外體可直接參與細胞間訊息的傳遞，其攜帶的核酸和蛋白質涉及影響受體細胞的生理狀態，並且與許多疾病的發生密切相關。然而，發展至今的技術尚未能有效率且自動化的分離胞外體和脂蛋白。目前主要有五類能從樣品中分離出胞外體的方法，即超高速差速離心法(differential ultracentrifugation)、超過濾法(ultrafiltration)、粒徑篩析層析法(size exclusion chromatography)、沉澱法(precipitation)、免疫親和法，其分別利用了胞外體的物理特性，如密度、大小以及表面抗原等生物特性，但這些方法往往面臨處理樣品耗時長、樣品處理通量低、純化程度不高、以及改變或損傷胞外體等問題，進而影響了後續研究發展及臨床應用。因此，本研究目標是開發出利用微流體裝置模組以分離胞外體，其可連續式獨立操作且可因應需求調整分離條件。在本研究當中，已成功利用溫度梯度製備出具有不同孔徑大小的凝膠，並建立不同粒徑螢光微珠的電泳實驗操作條件。相信此微流體裝置的建立能使往後的胞外體與脂蛋白研究更為精確且使用上更加便利。

Extracellular vesicles (EVs), secreted by cells to the outside of the cell and covered by bilayer phospholipids, have gradually been identified as one of the important mediators of cell communication. EVs have been explored as potential biomarkers for many diseases. However, the size of EVs is not only small but also widely distributed. The diameter of the EVs can range from 30 to 5,000 nm, and its molecular composition also varies depending on the type and the status of the secreting cell. In recent years, more and more studies have pointed out that EVs can directly participate in the transmission of cell-to-cell signals, and the cargo, including nucleic acids and proteins of EVs will affect the physiological state of the recipient cell and are closely related to many diseases. Currently, there are five main methods for the isolation of EVs from samples, including differential ultracentrifugation, ultrafiltration, size exclusion chromatography, precipitation, and immuno-affinity capture. These methods isolate EVs utilizing their physical biological characteristics, such as density, size, and surface antigens. But these methods are often faced with problems, such as the long processing time, low throughput, low purity, introducing modification or damages to EVs, all of which affect and limit the subsequent research and clinical applications as well. In addition, current technologies have not been able to efficiently and automatically isolate and separate EVs and lipoproteins due to their overlapped size and density. Hence, we aim to develop a microfluidic module to separate small-size EVs, which can be operated continuously and can be adjusted to achieve different separation conditions. We have successfully fabricated agarose gel of varying averaged pore size using temperature gradients. Polystyrene beads and EVs can be electrophoresed differentially based on their size and charges. It is believed that the establishment of this microfluidic device can facilitate the study of EVs and lipoproteins in the future.

摘要........................................I
Abstract....................................II
致謝.........................................IV
Table of Contents............................V
List of Figures..............................VIII
List of Tables...............................X
Chapter 1 緒論................................1
1.1 研究背景...................................1
1.2 胞外體 ( Extracellular vesicles, EVs ).....2
1.3分選胞外體的一般方法..........................3
1.4微流體技術 ( Microfluidics ).................4
1.5凝膠電泳 ( Gel electrophoresis ).............4
1.6梯度凝膠 ( Gradient gel )....................5
1.7凝膠種類.....................................6
1.8 脈衝場凝膠電泳 (pulsed-field gel electrophoresis，PFGE)........8
1.9 Continuous microfluidic assortment of interactive ligands (CMAIL)......................9
1.10 熱電致冷晶片(Thermoelectric cooler, TE cooler)...............9
1.11 掃描式電子顯微鏡 (Scanning Electron Microscope, SEM).........11
1.12 研究動機與願景...............................................11
Chapter 2 實驗設計與方法..........................................12
2.1. 微流道晶片裝置............................................13
2.1.1 溫度控制模組建立............................................13
2.1.2 壓克力母模雛形設計製作.......................................14
2.1.3 壓克力母模微流道裝置.........................................16
2.1.4 溫度梯度瓊脂糖凝膠 (agarose gel) 製備及翻模...................17
2.2. 梯度凝膠孔徑拍攝.............................................18
2.2.1 凝膠於SEM拍攝前處理.........................................19
2.3. 螢光微珠凝膠電泳實驗..........................................20
2.3.1 螢光微珠樣品................................................20
2.3.2 凝膠電泳架設................................................20
2.3.3 雙向凝膠電泳架設............................................21
2.4 細胞培養與胞外體收集...........................................22
2.4.1 人類胚胎腎細胞(HEK 293T)培養步驟.............................22
2.4.2 HEK 293T胞外體收集.........................................23
2.4.3 以NTA分析HEK 293T..........................................23
2.5. 實驗結果分析工具與方法........................................24
2.5.1 掃描式電子顯微鏡 (Scanning Electron Microscope, SEM)........24
2.5.2 倒立光學顯微鏡 (DMIL LED, Leica, Major, Germany)............24
2.5.3 倒立式電控顯微鏡系統 (DMI6000 B, Leica, Major, Germany)......24
2.5.4 Image J 生物影像處理分析軟體.................................24
Chapter 3 實驗結果與討論..........................................25
3.1 初步測試溫度梯度凝膠電泳結果....................................25
3.1.1 傳統製膠盤溫度梯度凝膠製備...................................25
3.1.2 凝膠降溫速度分析............................................26
3.1.3 螢光微珠凝膠電泳結果.........................................26
3.2. 螢光微珠凝膠電泳結果..........................................27
3.2.1 FCDG001 (60 nm)螢光微珠凝膠電泳結果..........................27
3.2.2 F8797 (97nm)螢光微珠凝膠電泳結果.............................32
3.2.3 60 nm及97 nm螢光微珠混合液凝膠電泳結果........................38
3.3. 溫度控制模組之溫差結果........................................42
3.3.1 以IRT拍攝凝膠形成過程.......................................42
3.3.2 以thermocouple量測並分析其降溫速率...........................44
3.4. SEM拍攝凝膠孔徑結果..........................................45
3.5 螢光微珠梯度凝膠電泳結果.......................................49
3.6 螢光微珠進行連續性梯度凝膠電泳結果..............................51
3.7 HEK 293T胞外體進行連續性梯度凝膠電泳結果........................54
Chapter 4 結論...................................................58
Chapter 5 未來展望...............................................60
References......................................................61

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