胞外泌體(extracellular vesicles, EVs)為細胞分泌的奈米級脂雙層囊泡，為細胞間距離溝通之工具，並且能夠攜帶DNA、RNA與蛋白質。受體細胞會以胞吞(endocytosis)、融合(fusion)、巨胞飲作用 (macropinocytosis)或是吞噬作用(phagocytosis)等等方式接收EVs與其攜帶的蛋白質與遺傳物質1。為了研究其所攜帶的蛋白質，科學家利用同量異位素標定，如 TMT (tandem mass tag) 2 或是以同位素標定，如SILAC (Stable Isotope Labeling with Amino Acids in Cell Cultures)3。然而2013年Alice Y. Ting團隊發表的APEX2對於標靶蛋白質體學有所突破，利用APEX2 活化生物素酚 (biotin phenol) 標定附近蛋白質，再以鏈霉抗生物素蛋白下拉(streptavidin pull-down) 方法純化感興趣蛋白作為後續質譜分析的樣品4,5。上述的方法大幅降低了雜訊與背景值，並且APEX2已成功對細胞內粒腺體的膜間隙內蛋白質進行標定5。而我們將APEX2與生物冷光共振能量傳遞系統 (bioluminescence resonance energy transfer, BRET) GFP-NanoLuciferase (GpNluc)結合，欲達成同時追蹤EVs與標定感興趣蛋白的多功能報導系統。而透過棕櫚醯化與PDGFR穿膜域將報導系統鑲嵌於膜外與膜內，我們期望以不同位點的報導系統了解EVs的蛋白質體學，並進一步解析細胞間透過EVs溝通之機制與參與蛋白。

Extracellular vesicles (EVs) are a type of cell-to-cell communication. They are nanosized vesicles with a lipid bilayer, containing DNAs, RNAs, and proteins from the donor cells1. Neighboring and distant recipient cells can take up EVs to facilitate the horizontal transfer of the genetic materials and proteins through endocytosis, fusion, macropinocytosis, and phagocytosis. In order to study the EV proteomics, scientist commonly use isobaric labeling like TMT (tandem mass tag) 2, as well as isotopic labeling such as SILAC (Stable Isotope Labeling with Amino Acids in Cell Cultures)3, to target proteins of interest (POI)。On the other hand, APEX2, an engineered peroxidase, biotinylates nearby protein by generating biotin phenol radical. The biotinylated proteins are further purified by streptavidin pull-down assay for mass spectrometry4,5. This method not only decreases the background from non-biotinylated protein but also increases the purity of POI. Alice et al. demonstrated APEX2 on the inner membrane of mitochondria to target proteins in the interspace of mitochondria5. In this study, we combined our BRET (bioluminescence resonance energy transfer) EV imaging system, PalmGpNluc with APEX2 for EV imaging while achieving proximal-dependent biotinylation and EM (electron microscopy) imaging. By fusing GpNluc, APEX2 with S-palmitoylation or PDGFR transmembrane domain (PDGFR-TM), we successfully labeled our multifunctional reporter system to the inner or outer membrane of EVs, respectively, thereby enabling EV imaging and proteomics analysis to elucidate mechanisms of EV-mediated intercellular communication.

中文摘要 ii
Abstract iv
致謝 v
縮寫表 vi
文目錄 ix
圖目錄 xii
第一章 文獻探討 1
1-1.胞外泌體 (Extracellular Vesicles, EVs) 1
1-2. 生物冷光 (Bioluminescence, BL) 5
1-3. 生物冷光共振能量轉移 (Bioluminescence Resonance energy transfer, BRET) 7
1-4. 嵌膜蛋白(Membrane Anchored Protein) 8
1-4-1. S-棕櫚醯化 (S-Palmitoylation) 8
1-4-2. 血小板衍生生長因子受體穿膜區 (platelet-derived growth factor receptor transmembrane domain, PDGFR-TM) 10
1-5. 胞外泌體的蛋白質體學 11
1-5-1. Nano Liquid chromatography-(tandem) mass spectrometry (nLC-MS/MS) 11
1-5-2. Screening and Targeted EV Proteomics 12
1-5-3. MS/MS Data Acquisition 12
1-5-4. 特定篩選反應監測Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM) 14
1-5-5. 平行反應監測(Parallel Reaction Monitoring) 15
1-6. 蛋白質標定技術 15
第二章 研究動機 18
第三章 實驗材料與操作方法 20
3-1. 實驗材料 20
3-1-1. 化學藥品與材料 20
3-1-2. 儀器 25
3-2. 實驗方法 27
3-2-1. 質體去氧核醣核酸(Plasmid DNA)建構 27
3-2-2. 細菌形質轉換(Bacteria Transformation) 29
3-2-3. 細胞株培養 30
3-2-4. 胞外泌體分離 30
3-2-5. Nano-Glo 螢光素酶試驗 (Nano-Glo Luciferase Assay) 31
3-2-6. 細胞生物素化標定 31
3-2-7. 西方點墨法(Western Blot Analysis) 32
3-2-8. 胞外泌體墨點分析 34
3-2-9. 胞外泌體生物素化標定 34
3-2-10. 鏈霉抗生物素蛋白下拉方法純化生物素化標定蛋白 36
3-2-11. 銀染定量與影像紀錄 36
第四章 實驗結果 38
4-1質體建構 38
4-2. 細胞螢光觀測與生物冷光能量共振(BRET)測試 41
4-3細胞生物素標定測試 44
4-4. 胞外泌體的螢光觀測與生物冷光共振 46
4-5確認報導系統於EVs膜內外之測試 48
4-6胞外泌體生物素標定測試 50
4-7分泌信號 (secretion signal) 添加增強報導蛋白穿膜表現 54
4-8 ssAGTM與ssGATM於細胞生物素化標定測試 56
4-9 ssAGTM之胞外泌體定性測試 58
第五章、結果討論 60
第六章、 參考文獻 64

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