胞外泌體是由細胞所分泌的奈米級脂雙層囊泡，除了可作為一種細胞之間聯繫的媒介之外，還可以伴隨著胞吞(endocytosis)的機制來裝載及運送有機分子。因此，了解並觀察胞外泌體的動態分佈可以幫助科學家釐清生命現象的機制，例如癌症轉移、血管新生等等。
近十年以來，科學家發現生物冷光在非侵入式分子影像領域中擁有低背景雜訊和高靈敏度的優點。其中，一種新的冷光系統 NanoLuc (Nluc)，相較於往常傳統冷光(e.g., Renilla luciferase (Rluc), Firefly luciferase (Fluc))有更卓越的表現，其具有半衰期大於兩小時的發光時間以及發光強度約為Rluc、Fluc強度的150倍。除此之外，生物冷光共振能量轉移在分生研究領域也佔有一席之地，其優點是使用自身發出的冷光當作光源，與螢光共振能量轉移是使用外來激發光當作光源相比，光漂白及自發螢光這些缺點也獲得改善，因此適合用於生醫影像。
本篇論文使用多功能報導蛋白，LSSmOrange Nluc (OgNluc) 及palmitoylated LSSmOrange Nluc (PalmOgNluc)，來標靶胞外泌體以得到影像實體化及追蹤胞外泌體用途。在細胞實驗上，證實棕櫚酰化並不會對螢光蛋白造成影響。此外，在4小時時間的Nluc活性檢測試驗中，相較於OgNluc，PalmOgNluc在細胞上有較穩定的訊號表現；在胞外泌體上，冷光活性表現強度較強。在未來動物實驗，希望在帶有PalmOgNluc的胞外泌體在深層細胞內可以達到良好的影像效果，達到時間與空間上的精確性。

For multicellular organisms, the connection between the cells is important in maintaining the physiological homeostasis of life. Recently, scientists have found that cells can exchange information by extracellular vesicles (EVs). EVs are nanoscale vesicles secreted by the cells. In addition to being as a medium of intercellular communication, EVs can also load and transport molecules via endocytosis, fusion and receptor-uptake, etc. Therefore, understanding and observing the dynamic distribution of EVs may help scientists understand and clarify the mechanisms of EV-mediated biological phenomena, such as cancer progression and metastasis.
Over the past decade, scientists have found that bioluminescence, in the field of non-invasive molecular imaging, has the advantages of providing a low background noise ratio and high signal intensity. Hence, bioluminescence imaging technology has increasingly been used in preclinical biomedical researches. Recently, a new bioluminescence system, NanoLuc (Nluc), has been developed. Nluc is superior to conventional luciferases, such as Rluc and Fluc. Nluc’s signal is 150-fold higher than that of Rluc and Fluc. Besides, bioluminescence resonance energy transfer (BRET) has a place in biomedical research. Comparing to fluorescence resonance energy transfer, without the need of excitation source, photobleaching and autofluorescence can be mitigated in BRET, which is suitable for biomedical imaging.
In this thesis, we engineered optical reporters, LSSmOrange Nluc (OgNluc) and palmitoylated LSSmOrange Nluc (PalmOgNluc), labeling EV populations for visualization and tracking EVs release. In vitro assay, we confirm that palmitoylation does not influence the function of fusion protein. In addition, in the 4-hour detection for Nluc activity, we found that compared with OgNluc, PalmOgNluc has better stability in cells and better signal in EVs. In future, we hope that EV-PalmOgNluc has a better resolution in the deep tissue for in vivo with accurate spatiotemporal resolution.

摘要 i
Abstract ii
致謝 iv
縮寫表 vi
目錄 vii
一. 文獻回顧與探討 1
(一) 胞外泌體 (Extracellular Vesicles; EVs) 1
(二) 生物冷光 (Bioluminescence, BL) 3
(三) 生物冷光共振能量轉移 (Bioluminescence Resonance Energy Transfer, BRET) 5
(四) S-棕櫚酰化 (S-Palmitoylation) 7
(五) 研究動機 9
二. 實驗材料與操作方法 10
2.1 材料 10
2.2 儀器 13
2.3 操作方法 14
2.3.1 報導系統之建立 (Construction of EV-BRET System) 14
2.3.2 膠體電泳分析 (Gel Electrophoresis Analysis) 15
2.3.3 細胞培養 (Cell Culture) 16
2.3.4 細胞轉染 (Transient Transfection) 16
2.3.5 慢病毒載體之建立 (Construction of Lentiviral Particles) 17
2.3.6 報導基因轉導至細胞內 (Transduction of the Reporter Gene into HEK 293T Cells) 17
2.3.7 活細胞共軛焦顯微鏡觀測 (Live-cell Confocal Microscopy) 17
2.3.8 奈米粒子追蹤分析 (Nanoparticle Tracking Analysis, NTA) 18
2.3.9 胞外泌體生產及分離 (EV Production and Isolation) 18
2.3.10 點墨法 (Dot Blot Analysis) 19
2.3.11 蔗糖密度梯度離心 (Sucrose Density Gradient) 20
2.3.12 西方墨點法 (Western Blot) 20
2.3.13 Nano-Glo 螢光素酶試驗 (Nano-Glo Luciferase Assay) 21
2.3.14 細胞中生物冷光影像 (BLI Image in vitro) 21
三. 實驗結果 22
3.1 膠體電泳分析 22
3.2 報導蛋白在細胞中的表現 24
3.3 細胞之生物冷光表現 25
3.4 細胞溶解產物之西方墨點法分析 27
3.5 奈米粒子追蹤分析 29
3.6 點墨法 31
3.7 PalmOgNluc標靶不同族群之胞外泌體 33
3.8 蔗糖梯度離心 35
3.9 四小時冷光活性及穩定性檢測 37
四. 討論 39
五. 結論 41
六. 參考文獻： 42

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