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姓名	陳學田(Hsueh-Tien Chen)  查詢紙本館藏	畢業系所	化學學系
論文名稱	開發以巨大細胞膜囊泡為基礎之自支撐仿生膜平台 (Development of free-standing in vitro membrane platform based on giant plasma membrane vesicles)
檔案	[Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 (2024-8-31以後開放)
摘要(中)	具備穩定性與高通量的仿生膜為探索細胞膜上生物分子的動力學研究提供了優秀的平臺。其中，利用人造微脂體的囊泡融合便能輕鬆取得的支撐式仿生膜是最廣為使用的仿生膜平台之一。但是支撐式膜平台中基材對於脂雙層的影響以及組成複雜性不足以模擬行使著複雜生理功能的細胞膜。而利用來自細胞的巨大細胞膜囊泡 (giant plasma membrane vesicles, GPMV) 建構仿生膜平台，則能夠保留來自細胞膜的脂質與蛋白質組成。本研究嘗試整合GPMV以及具孔洞的微流體設計，建構具有天然膜蛋白的自支撐脂雙層 (free-standing lipid bilayer)，以期能夠保留仿生膜平台的生理複雜性，並利用孔洞設計使其達到自支撐的特性，進而改善蛋白質等特定研究標的受到基材影響的問題，從而提升仿生膜實驗設計的自由度與生物應用性。此論文利用影像分析對基礎實驗架構進行了定量統計，包含了微流體孔洞製程、溫度對GPMV大小與產率的影響、微流體中GPMV的攔截效率等。進一步的實驗則嘗試調整了溫度與滲透壓等物理參數，進而使得原來因富含膽固醇而難以破裂的GPMV在微流體中可以達到高通量的破裂並成膜。GPMV成膜後則利用光漂白螢光回復實驗與共軛焦螢光顯微鏡對建構的仿生膜平台對膜的二維流動性與自支撐性質進行鑑定，並針對此仿生膜平台尚未達成的自支撐特性進行囊括幾何結構與表面性質的調整。
摘要(英)	In vitro membrane featuring good stability and high throughput capability can be a useful platform for the investigation of biomolecular interaction and kinetics on the cell membrane. Supported lipid bilayer (SLB), which can be prepared by fusion of artificial liposomes, is one of the most widely used biomimetic membrane platforms. However, the interference from the underlying solid support and the difficult in recapitulating cell membrane compositions suggest that SLB may have limited capacity to simulate cell membrane that performs sophisticated functions. To address such limitations, giant plasma membrane vesicles (GPMV), which retain the native cell membrane compositions, have been used as alternative membrane source, for constructing in vitro membrane. This work aimed to integrate GPMV and micro-patterned substrate in microfluidics chip to construct free-standing lipid bilayer with native membrane protein, and hoped to improve the applicability of biomimetic membrane system. Quantitative image analysis was applied to characterize the micro-patterned substrate, GPMV size, temperature dependency of GPMV production and vesicle-capturing efficiency. Furthermore, experiments were performed to optimize physical parameters such as the temperature and osmotic pressure, that can result in efficient GPMV rupture to form the lipid bilayer. Fluorescence recovery after photobleaching (FRAP) and confocal microscope were used to study the lateral diffusion and free-standing properties of the resulting lipid membrane, Additional effort that are necessary to form free-standing lipid bilayer platform directly from GPMV was discussed.
關鍵字(中)	★ 仿生膜平台 ★ 巨大細胞膜囊泡 ★ 自支撐式脂雙層 ★ 囊泡破裂	關鍵字(英)	★ biomimetic membrane platform ★ giant plasma membrane vesicle ★ free-standing lipid bilayer ★ vesicle rupture
論文目次	國立中央大學圖書館學位論文授權書 i 指導教授推薦書 ii 口試委員審定書 iv 摘要 v Abstract vi 致謝 vii 目錄 viii 圖目錄 xi 一、緒　論 1 1-1　　生物細胞膜之基礎性質 1 1-1-1　　流體鑲嵌模型 (fluid mosaic model) 1 1-1-2　　脂筏假說 (lipid raft hypothesis) 2 1-2　　細胞膜成分之功能性研究與困難 2 1-3　　仿生膜平台的發展 3 1-4　　細胞外囊泡與巨大細胞膜囊泡 4 1-4-1　細胞外囊泡 5 1-4-2　巨大細胞膜囊泡 5 1-5　　自支撐式膜平台 6 1-6　　囊泡破裂形成脂雙層之機制 7 1-7　　研究目的 8 二、實 驗 材 料 與 方 法 10 2-1　　藥品與材料 10 2-1-1　光蝕刻與微流體晶片製備 10 2-1-2　細胞培養 10 2-1-3　巨大細胞膜囊泡之製備 10 2-1-4　CYTOP表面改質 11 2-2　　實驗儀器 11 2-3　　晶圓製作 12 2-4　　微流體晶片製作 12 2-4-1　捕獲層之製作 13 2-4-2　孔洞層之製作 14 2-5　　細胞培養 15 2-6　　巨大細胞膜囊泡 (GPMV) 製備 16 2-7　　建立與優化囊泡之成膜條件 17 2-8　　螢光影像與光漂白螢光恢復實驗 (Fluorescence recovery after photobleaching, FRAP) 18 2-9　　共軛焦螢光影像 18 2-10　　CYTOP表面改質 19 三、實　驗　結　果　與　討　論 20 3-1　　微流道晶片設計 20 3-1-1　捕獲層設計 20 3-1-2　孔洞層設計 22 3-1-3　孔洞層孔徑影像定量 23 3-2　　巨大細胞膜囊泡 (GPMV) 25 3-2-1　具螢光蛋白標記的細胞膜與GPMV 25 3-2-2　GPMV生成時之溫度效應 27 3-3　　捕獲層捕獲效率 29 3-3-1　捕獲層實時捕獲率 29 3-3-2　捕獲層理想捕獲率 31 3-4　　巨大細胞膜囊泡破裂形成之仿生膜平台 33 3-4-1　以溫度與親水性表面達成GPMV破裂與成膜 33 3-4-2　嘗試引入滲透壓環境改變增加GPMV破裂機率 34 3-5　　以FRAP鑑定囊泡破裂成膜後的流動性 37 3-6　　以共軛焦顯微鏡鑑定成膜於孔洞層後是否實現自支撐特性 38 四、後　續　工　作　與　展　望 40 4-1　改善物理條件以追求自支撐可能性 40 4-1-1　降低孔洞層高度 40 4-1-2　以CYTOP進行表面改質 41 4-2　　結論 43 4-3　　未來展望 43 參考文獻 45
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指導教授	涂熊林 謝發坤(Hsiung-Lin Tu Fa-Kuen Shieh)	審核日期	2021-8-30
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