胞外泌體從細胞產生，可以用來與相鄰細胞或遠處細胞溝通，胞外泌體係一含有跨膜蛋白的脂雙層結構，內含細胞質內的蛋白、核醣核酸、去氧核醣核酸及脂質；根據細胞狀態，會分泌不同種類胞外泌體。現今，胞外泌體影像面臨一些挑戰，如：微小的胞外泌體成像，以及在細胞及活體之間，觀察胞外泌體釋放及運送的動態，為了克服這些問題，觀察胞外泌體的影像需仰賴特定的報導基因，以及健全的活細胞分子影像技術做為輔助；在此，我們使用一個光學的報導基因去標定胞外泌體，優化胞外泌體追蹤影像；我們融合了增強綠色螢光蛋白(enhanced fluorescence protein; eGFP) 和Nanoluc® (Nluc) 冷光素酶，以及參考過去棕櫚酰化的基因片段PalmGFP，精確標定於細胞膜內，使胞外泌體產出時帶有更高比率之報導基因蛋白。藉由基質furimazine的利用，產生的冷光經由生物發光共振能量轉移（bioluminescence resonance energy transfer, BRET）激發螢光，使報導蛋白兼具有螢光強度的優勢及冷光訊號雜訊比低的長處，發展高檢測靈敏度與長時間穩定的光源，作為研究胞外泌體分子影像的工具。
在本篇研究指出，棕櫚酰化的生物發光共振能量轉移的報導蛋白，可運用在胞外泌體及細胞，是一個創新且優化標定胞外泌體的工具，在研究胞外泌體上，希望可以克服在體內之胞外泌體觀測深度、動態胞外泌體釋放及交換情形；藉由此嶄新之報導基因蛋白，搭配高解析度的共軛焦顯微鏡技術，可以在細胞和胞外泌體兩者作為即時觀測使用。

Cells can communicate with neighboring and distant cells via extracellular vesicles (EVs). EVs are lipid bilayered structures composed of cytosolic proteins, RNAs, DNAs and lipids. Cells secrete different types of EVs from different sub-cellular origin. Recently, EV imaging needs optimal reporter to visualize EVs subpopulations, observing specific labelled EVs in vitro and in vivo and dynamics of EV biogenesis. Due to these reasons, EVs delivery requires specific reporters and robust live-cell imaging technology. Here we create an optical reporter to label EVs for optimal tracking of EVs utilizing eGFP fused with Nluc sequence and palmitoylation sequence. Palmitoylation can anchor a reporter to the inner leaflet of a lipid bilayer. Using its substrate furimazine, bioluminescence light produces and excites fluorescent light via bioluminescence resonance energy transfer (BRET). Fluorescence emits higher intensity light and bioluminescence offers better signal-to-noise ratio. This reporter provides a long-term and brighter light for studying EVs.
In this study, GpNluc and PalmGpNluc reporter protein can be applied in both cells and EVs. We believed that these advantages combined together is an optimal tool can be applied in EV tracking and visualization. Acting as an imaging platform for both in vitro and in vivo compare with other state-of-the-art EV labeling methods.

摘要 i
Abstract ii
致謝 iii
縮寫表 v
目錄 vii
第一章 前言 1
1.1 細胞溝通途徑發展史 1
1.2 胞外泌體(Extracellular Vesicles) 2
1.2.1 外泌小體(Exosomes) 4
1.2.2 微泌體(Microvesicles) 5
1.2.3凋亡小體(Apoptotic Bodies) 6
1.2.4溶瘤體(Oncosomes) 6
1.3 增強綠色螢光蛋白(Enhanced Green Fluorescent Protein) 7
1.4 生物冷光蛋白(Bioluminescent Protein) 8
1.5 生物發光共振能量轉移(Bioluminescent Resonance Energy Transfer, BRET) 9
1.6 棕櫚酰化 (Palmitoylation) 10
1.7 胞外泌體標定技術(Extracellular Vesicles Labeling Methods) 11
1.8 研究目的及目標 12
第二章 材料及方法 13
2.1材料 13
2.2研究方法 16
2.2.1報導蛋白構造製作 16
2.2.2 聚乙烯亞胺轉染(Transient Mammalian Cell Transfection with Polyethylenimine (PEI)) 20
2.2.3慢病毒轉染生產(Lentiviral Production Infection) 20
2.2.4細胞培養(Cell Culture) 21
2.2.5 胞外泌體生產及離心 21
2.2.6 BCA蛋白質定量 22
2.2.7 DNA點墨免疫學活性測定(Dot Blot Analysis) 22
2.2.8西方點墨法分析(Western Blot Analysis) 23
Bio-Rad 系統 23
Invitrogen 系統 24
2.2.9蔗糖梯度離心法 26
2.2.10 冷光及生物發光共振能量轉移測定方法 28
2.2.11 奈米粒子追蹤分析 28
2.2.12 細胞存活率與細胞毒性試驗 29
第三章 結果 30
3.1報導蛋白質體定性 30
3.1.1 pDEST-GpNluc質體定性 30
3.1.2 pDEST-PalmGpNluc質體定性 32
3.1.3 pDEST-GpNlucPalm質體定性 34
3.2報導蛋白質體於HEK293T細胞內表現 37
3.2.1報導蛋白在共軛焦顯微鏡表現 37
3.3 DNA斑點染漬免疫學活性測定(Dot Blot Analysis) 40
3.4 活細胞Furimazine使用及觀測表現 43
3.5 BRET細胞基質活性測試 44
3.6 BRET胞外泌體基質活性測試 46
3.7 奈米粒子追蹤分析儀(Nanoparticles Tracking Analysis) 48
3.8 PalmGpNluc標定不同過濾法之胞外泌體 50
3.9 細胞殘骸西方點墨法分析 51
3.10 胞外泌體冷光訊號偵測及西方點墨法結果比較 52
第四章 討論 54
第五章 結論 59
參考資料 60

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