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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林啟萬(Chii-Wann Lin) | |
dc.contributor.author | Hui-Wen Liu | en |
dc.contributor.author | 劉惠文 | zh_TW |
dc.date.accessioned | 2021-06-08T00:02:10Z | - |
dc.date.copyright | 2020-09-17 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-06 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17232 | - |
dc.description.abstract | 近年來,癌症胞泌體(Exosome)被發現與癌症細胞轉移(Metastasis)有密切相關,其癌細胞在從原位癌轉移至其他器官之前,會先分泌帶有靶向性蛋白之胞泌體至欲轉移之器官建立腫瘤微環境(pre-metastatic niche),因此若是能在癌細胞轉移前早期發現,可望為更有效地癌症治療帶來一線生機。因此本研究欲利用結構最佳化的核酸適體(Aptamer)作為探針,結合高靈敏度且免標記的相位式表面電漿子共振感測器(Phase-sensitive Surface Plasmon Resonance Biosensor, pSPR biosensor)進行肺臟靶向性之乳癌細胞(4175-LuT)之胞泌體膜蛋白α6β4整合素蛋白(Integrin)之檢測。 一般核酸適體是經由SELEX(Systematic evolution of ligands by exponential enrichment)所選殖而得。然而經由SELEX選出的核酸適體存在著潛在的缺點:包含前後引子可能參與目標分子的結合反應或PCR步驟傾向排除二級結構導致擴增反應的偏誤等,導致該適體結構並非為最佳設計。另一方面,若是可以精簡化SELEX篩選出來的適體序列,即可根據目標情境的需求不同,進一步結合其他核酸適體序列或是加入人工序列,設計具有複合功能之適體。 為使適體結構最佳化,我們使用核酸二級結構模擬工具及序列相似度分析工具,推測出合理之適體共識區域後進行裁切或是延長數個鹼基以維持重要二級結構,設計出21個鹼基的α6β4適體IDA3t,並經由SPR感測器即時檢測核酸適體與目標分子的結合情形,同時在掃描式電子顯微鏡底下觀察其形貌及進行顆粒數統計。另外,我們進一步設計了一個含有一段人工序列的適體IDAχ,並透過實驗證明其仍保有對目標分子的專一性。此人工適體預期可以在捕捉目標分子之後進一步釋放與觸發核酸雜合連鎖反應(Hybridized Chain Reaction, HCR)有關的序列,達到放大訊號的功能,以期未來可以達到在血液樣品當中專一性檢測的目標。 | zh_TW |
dc.description.abstract | Recently, tumor exosome is discovered to play a vital role in cancer metastasis process, that the integrins on the surface of tumor exosome would determine organtropic metastasis. Tumor-derived exosome would be uptaken by organ-specific cells and prepare the pre-metastatic niche before metastasis happened. If we can detect these exosomes in advance, it’s expected to bring other possible approaches to cancer treatment. In this work, we demonstrate the development of metastatic exosome sensing strategies using Phase-sensitive Surface Plasmon Resonance (pSPR) as a platform, which is highly sensitive and label-free, for aptamer structural rationalization. 4175 lung-tropic tumor cell derive exosome (4175-LuT) is studied with surface integrin α6β4 as targeted surface marker. Traditionally, aptamers are made by SELEX(Systematic evolution of ligands by exponential enrichment). However, there’s some sequence-structural bias with it. For instance, the primers might also attend in the binding with target molecules, or PCR (Polymerase chain reaction) is preferred to amplify the DNA sequence that is lack of secondary structure, while aptamers usually bind with target molecules via it. On the other hand, we can design a bifunctional aptamer based on structural rationalization to meet the need of sensing different target molecules. In order to optimize the structure of α6β4 aptamer made by SELEX, we use secondary structure simulation tools (NUPACK, Mfold) and similarity analysis tool (Lalign) to analyze the structure of aptamer, the optimized α6β4 aptamer named IDA3t. We confirm the binding affinity between optimized aptamer and target molecule by pSPR biosensor. Also, we observe the exosome morphology through Scanning Electron Microscopy (SEM), do particle size analysis and particle number statistics through the images. Furthermore, we design an artificial aptamer here, named IDAχ, which is combined with 21 mer IDA3t and 22 mer designed sequence, and we demonstrate that it can capture 4175-LuT exosome still. This “IDAχ” aptamer has tailor made hairpin secondary structure that can be developed into more elaborated molecular machinery such as hybridization chain reaction, for further application of detecting target molecules with clinical samples. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:02:10Z (GMT). No. of bitstreams: 1 U0001-0608202016582700.pdf: 3705365 bytes, checksum: 55982eec086d2b5d47fe84aab0fbf54a (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 誌 謝 I 摘 要 II Abstract III 目 錄 V 圖目錄 VII 表目錄 IX 第一章 緒論 1 1.1 研究背景 1 1.2 適體結構設計方法之提出 2 1.3 章節架構 3 第二章 基本原理與文獻回顧 4 2.1 SELEX基本原理與核酸適體簡介 4 2.1.1 SELEX技術原理 4 2.1.2 核酸適體特性 5 2.1.3 SELEX限制及核酸適體篩選後修飾(Post-SELEX Modifications) 5 2.2 表面電漿子共振生物感測器 6 2.2.1 表面電漿子共振原理及激發方式 7 2.2.2 強度式表面電漿子共振 10 2.2.3 相位式表面電漿子共振 11 2.2.4 表面化學修飾方式 12 2.3 癌症細胞胞泌體(Exosome) 13 2.3.1 胞泌體介紹 13 2.3.2 帶有指向性的轉移性癌細胞前驅物 14 2.4 適體與目標分子的作用方式 16 2.5 雙重功能適體 18 第三章 研究方法與材料 19 3.1 晶片製程暨流道製作 19 3.1.1 基材清洗 20 3.1.2 物理氣相蒸鍍 20 3.2 相位式全像式表面電漿子共振感測器 21 3.2.1 光學系統架構 21 3.2.2 Labview量測介面 22 3.3 適體性質分析方法 23 3.3.1 動力學方式 23 3.3.2 熱力學方式 24 3.4 適體結構最佳化 24 3.4.1 核酸二級結構模擬工具 25 3.4.2 核酸序列相似度分析工具 27 3.4.3 適體結構最佳化後之延伸設計 28 3.5 掃描式電子顯微鏡(Scanning Electron Microscope, SEM) 29 3.5.1 掃描式電子顯微鏡基本原理 29 3.5.2 實驗使用參數及試片準備流程 30 第四章 研究結果與討論 32 4.1 最佳化適體設計結果及表面適體修飾 32 4.2 表面電漿子共振感測器捕捉胞泌體表面整合素蛋白 34 4.3 SEM影像 37 4.4 胞泌體性質分析 39 4.4.1 使用Imagej軟體分析 39 4.4.2 統計結果 40 4.5 Anti-CD9抗體結合力分析 41 第五章 結論與未來展望 43 第六章 參考文獻 45 | |
dc.language.iso | zh-TW | |
dc.title | 基於表面電漿子共振生物感測器之適體結構最佳化工程應用於偵測轉移性癌症胞泌體整合素蛋白 | zh_TW |
dc.title | Development of Metastatic Exosome Sensing Strategy via Surface Plasmon Resonance based Aptamer Structural Rationalization | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 沈湯龍(Tang-Long Shen),黃念祖(Nien-Tsu Huang) | |
dc.subject.keyword | 表面電漿子共振,適體,胞泌體,整合素, | zh_TW |
dc.subject.keyword | Surface Plasmon Resonance,Aptamer,Exosome,Integrin, | en |
dc.relation.page | 49 | |
dc.identifier.doi | 10.6342/NTU202002559 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2020-08-07 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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