指導教授：張煥正臺灣大學：化學研究所黃紫葳Huang, Tzu-WeiTzu-WeiHuang2014-11-252018-07-102014-11-252018-07-102014http://ntur.lib.ntu.edu.tw//handle/246246/261300腫瘤是由非勻相細胞所組成，其中有一群特定的腫瘤細胞具有類似幹細胞自我更新及不斷的增生出子代癌細胞的能力，即為癌症幹細胞(cancer stem cells；CSCs)。此群細胞被認為在癌症的形成、癌症復發及轉移、抗藥性有密不可分的關係。因此如何以螢光標記物標記追蹤此群癌症幹細胞成為重要的研究課題。 而螢光染料用於細胞標記雖具強螢光強度且標記專一等優點，然而螢光容易消失，而有著一定限制。而螢光奈米鑽石不僅有高生物相容性且有穩定的螢光性，克服了螢光染料不利於長時間的追蹤的缺點，因此設計了一新穎的策略來標記追蹤癌症幹細胞。 本論文中結合螢光奈米鑽石和乳癌細胞，利用螢光奈米鑽石的螢光判斷細胞的分化狀況、和觀察非黏附性乳腺球細胞(mammosphere)的成長，並和常用的細胞追蹤螢光染料CFSE (carboxyfluorescentsuccinimidy ester)及EdU (5-ethynyl-2-deoxyuridine)做比較。實驗一開始先由非黏附性乳腺球的重新生長實驗證實癌症幹細胞具自我更新能力，並以共軛焦顯微鏡和流式細胞儀觀察，發現在第二代的非黏附性乳腺球細胞中仍然可偵測到螢光奈米鑽石之螢光，而CFSE和EdU標記的細胞則無螢光維持。而由細胞追蹤20天實驗當中，比較三種螢光標記物追蹤具有癌症幹細胞特性之細胞的能力，結果顯示20天後螢光奈米鑽石相較於CFSE及EdU，可維持在非黏附性乳腺球細胞中有較高的螢光強度，表示螢光奈米鑽石在此實驗策略中是為長時間追蹤癌症幹細胞群的有利工具。Cells within tumor population exhibit functional and phenotypic heterogeneities, and there exists a minor population of tumor cells, termed cancer stem cells (CSCs), having the capacity of self-renewal and differentiation. These CSCs are thought to drive tumor growth, tumor recurrence, drug resistance and metastasis. Therefore, how to use markers to label and track CSCs has been an important issue in biomedical research. Although organic dyes have been commonly used for cell labeling due to their advantages of strong fluorescence emission and easiness of conjugation with biomolecules, but there are some restrictions, such as photobleaching, which prevent long-term observations. This problem can be readily overcome by using fluorescent nanodiamonds (FNDs), which have not only high biocompatibility but also excellent photostability. Here, we proposed to use the FND labeling method to investigate the cell differentiation and growth of primary mammospheres, with the results to be compared with those of the fluorescent markers, CFSE (carboxyfluorescentsuccinimidy ester) and EdU (5-ethynyl-2-deoxyuridine). The formation of primary mammospheres is a measure of stem cell activity, and thus cannot be used to assess stem cell self-renewal. However, primary mammospheres can be harvested, digested to single cells, and passaged for the assessment of their self-renewal (secondary generation). In this study, we found that mammospheres derived from ASB-145-1R cells can be passaged to satisfy the true criteria of self-renewal. The fluorescence of FND, but not of CFSE and EdU, can be detected in the second generation of the mammospheres by using confocal microscopy and flow cytometry. Results of long-term labeling and tracking of breast CSCs show that the number of cells retaining FNDs in the mammospheres is higher than those of CFSE- and EdU-labeled cells. It is concluded that the FND labeling is a valuable and effective tool for long-term tracking of CSCs.口試委員會審定書 # 中文摘要 i Abstract iii 目錄 v 圖目錄 viii 表目錄 x 第一章 緒論 1 1.1 前言 1 1.2 癌症幹細胞 1 1.2.1 幹細胞 1 1.2.2 癌症幹細胞特性 2 1.3 細胞標記(cell labeling)及細胞追蹤(cell tracking) 4 1.4 細胞追蹤應用 4 1.4.1 5-Ethynyl-2′-deoxyuridine (EdU) 6 1.4.2 Carboxyfluorescein diacetate succinimidyl ester (CFDASE, CFSE) 7 1.4.3 奈米材料應用於細胞標記 8 1.5 螢光奈米鑽石 12 1.5.1 鑽石的結構 12 1.5.2 鑽石的種類 13 1.5.3 螢光奈米鑽石的缺陷中心 14 1.5.4 螢光奈米鑽石的特性 15 1.6 研究動機 16 第二章 實驗儀器裝置與藥品 17 2.1 實驗儀器 17 2.1.1 氦離子束佈值裝置 18 2.1.2 流式細胞儀 20 2.2 實驗藥品 22 第三章 實驗步驟 24 3.1 製備粒徑為100奈米的螢光奈米鑽石 24 3.1.1 奈米鑽石的佈值前處理 24 3.1.2 離子束佈值 25 3.1.3 高溫淬火 26 3.2 利用螢光奈米鑽石追蹤乳腺癌幹細胞 27 3.2.1 乳癌癌症幹細胞來源 27 3.2.2 乳癌癌症細胞培養 27 3.2.3 非黏附性乳腺球(mammosphere)形成 27 3.2.4 EdU標記乳癌細胞 28 3.2.5 螢光奈米鑽石標記乳癌細胞 28 3.2.6 CFSE染色 28 3.2.7 非黏附性乳腺球細胞膜染色 28 3.2.8 非黏附性乳腺球分離成單顆細胞 29 3.2.9 實驗流程 29 第四章 實驗結果 30 4.1 非黏附性乳腺球重新生長(Mammosphere re-growth) 30 4.1.1 使用顯微鏡觀察非黏附性乳腺球大小、數量 31 4.1.2 用流式細胞儀測螢光強度 35 4.1.3 用共軛焦顯微鏡拍攝非黏附性乳腺球 38 4.2 乳癌幹細胞的20天長時間追蹤 40 第五章 結論 45 參考資料 464383248 bytesapplication/pdf論文公開時間：2017/07/04論文使用權限：同意有償授權(權利金給回饋學校)螢光奈米鑽石乳腺癌幹細胞細胞標記[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261300/1/ntu-103-R01223154-1.pdf