臺灣博碩士論文加值系統

螢光奈米鑽石 (fluorescent nanodiamond, FND)，是一種將type Ι鑽石以高能量離子束撞擊，並經高溫退火處理後，放出穩定紅色螢光的奈米材料。螢光奈米鑽石具有優異的光穩定性，可放出較佳組織穿透力的紅色螢光，再加上高度生物相容性與易於表面修飾的特性，螢光奈米鑽石應用在生物細胞研究上極具潛力。本研究係利用表面氧化過、直徑100奈米的螢光奈米鑽石做為細胞追蹤標記 (cell tracker)。細胞透過胞吞作用 (endocytosis) 攝取螢光奈米鑽石，並藉由流式細胞技術 (flow cytometry)分析標記的細胞樣品。另根據實驗細胞類型的不同，可將本論文區分成兩部分。
在第一部分研究中，利用螢光奈米鑽石標記脂肪前驅細胞株 (3T3-L1）與小鼠多分化潛能的骨髓基質幹細胞(489-2.1)，並以常用的細胞標記CFSE (carboxy-fluorescein diacetate succinimidyl ester)做為對照組。標記細胞的平均螢光強度會隨著時間而呈現近指數性 (exponentially) 衰退，由於螢光奈米鑽石的螢光極為穩定，可確定螢光強度的衰退是源自於細胞分裂。對於螢光奈米鑽石標記的細胞，從螢光衰退計算出來的螢光半衰期 (half-life)，與使用細胞記數法得到的細胞倍增時間 (cell doubling time) 非常接近，誤差小於10%；因此，藉由比較螢光奈米鑽石標記細胞的螢光強度，可以推估細胞經歷過幾次細胞分裂。此部分研究可以長時間追蹤細胞至第七次分裂。螢光奈米鑽石具有顯著的散射光增強效應，結合穩定的螢光性質，使其很適合搭配流式細胞分選儀 (fluorescence activated cell sorter, FACS) ，進行細胞分離篩選。本研究將螢光奈米鑽石標記的489-2.1細胞，從未標記的細胞中進行高純度分選。此外，細胞增殖測試 (cell proliferation assay) 的結果顯示，自螢光奈米鑽石標記細胞至細胞分選的整個流程，均未影響細胞的正常生長。
本論文於第二部分中，首度嘗試將螢光奈米鑽石應用於初代細胞 (primary cell)。於活體外 (in vitro)，利用螢光奈米鑽石標記自小鼠肺部組織直接萃取的肺部幹細胞(lung stem cell)，並透過靜脈內注射 (intravenous injection)，追蹤24小時內於成鼠體內 (in vivo) 標記的肺部幹細胞。經過流式細胞技術分析肺、肝、腎和脾組織的結果，只於肺有發現肺部幹細胞的訊號。同時藉由FACS，可分選出螢光奈米鑽石標記的肺部幹細胞。藉由顯微技術觀測肺部石蠟切片，發現肺部幹細胞僅分布於肺泡組織周圍及微支氣管末端。此外，免疫組織化學染色 (immunohistochemistry, IHC) 分析結果，更證明肺部幹細胞並未被巨噬細胞 (macrophage) 所吞噬。綜合實驗結果，本部分研究為肺部幹細胞的歸巢 (homing) 能力提供了一項有力的證據。
本研究首度嘗試建立一個利用螢光奈米鑽石標記初代幹細胞，並結合FACS與顯微技術來追蹤活體細胞的奈米平台。相信未來可望應用於活體內幹細胞長時間追蹤的相關研究。

Fluorescent nanodiamond (FND) is a sp3-carbon-based nanomaterial produced by radiation-damage followed by annealing. FND possess several unique properties such as excellent biocompatibility, facile surface modification, high tissue-penetrable red fluorescence and outstanding photostability, making it well suited for long-term labeling and tracking of cells. In this study, we use surface-oxidized FND particles (size~100nm) as the cell tracker. We introduce FNDs into cells by endocytosis through incubation, and do flow cytometric analysis. Based on different cell types, the thesis could be divided into two parts.
In the first part, we label 3T3-L1 pre-adipocytes and 489-2.1 multipotential stromal cells with FNDs, and use carboxy-fluorescein diacetate succinimidyl ester (CFSE) for comparison. Flow cytometric analysis reveals that the mean fluorescence intensity of the ingested FND particles per cell decays nearly exponentially with the number of cell division. The observed half-life matches well with the cell doubling time within 10%. Since the fluorescence emission of FNDs is perfectly stable, one can take advantage of this feature and determine conversely the number of generation that the cells have gone through by measuring their fluorescence intensity profile. In this study, we track to the seventh cell division. Also, we demonstrate for the first time that high-purity sorting of FND-labeled cells in a 1:1000 cell mixture is achievable by utilizing both the fluorescence and light scattering properties of this novel nanomaterial. Finally, we assessed the effect of the FND labeling and cell sorting on the 489-2.1 cells by using the cell proliferation assay. No significant difference in cell proliferation of FND-labeled cells before and after sorting was found. It signifies that fluorescence activated cell sorting (FACS) and the FND labeling do not alter the cell proliferation of the multipotential stromal cells.
In the second part, we demonstrate for the first time the application of FND to the primary cells. After labeling the lung stem cells directly isolated from mice with FNDs, we introduce those cells through intravenous injection. By flow cytometric analysis and microscopy, we find that the lung stem cells would reside particularly in the lung. We also use immunohistochemistry (IHC) to prove that the lung stem cells are not engulfed by macrophages. By combining the results in this study, we provide a convincing evidence that the lung stem cells do have homing ability.
In this study, we build up a biocompatible nanoparticle platform that combines FND labeling, FACS and microscopy. It had already begun to advance its ability to do long-term in vivo stem cell tracking research.

目 錄
誌謝……………………………………………………………………………………...I
中文摘要…………………………………………………………………….….….........II
Abstract…………………………………………………………………...……………IV
目錄…………………………………………………………………………………….VI
圖目錄…………………………………………………………………….……………IX
表目錄……………………………………………………………………………….....XI
第一章 緒論…………………………………………………………………………...1
第一節 研究動機…………………………………………………………………..1
第二節 幹細胞的介紹……………………………………………………………..1
第三節 肺部幹細胞 (lung stem cell )……………………………………………..3
第四節 初代細胞培養 (primary cell culture)……………………………………..5
第五節 螢光奈米鑽石 (fluorescence nanodiamond, FND)………………………5
第六節 螢光奈米鑽石的生物研究………………………………………………..7
第七節 螢光細胞標記……………………………………………………………..8
(一) 螢光有機染料…………………………………………………………….8
(二) 螢光蛋白………………………………………………………………….8
(三) 量子點…………………………………………………………………….9
第八節 奈米材料於幹細胞的研究………………………………………………10
第二章 實驗設計…………………………………………………………...............17
第三章 研究方法……...........……………………………………………………....18
第一節 材料....…………………………………………………………………...18
(一) 細胞來源………………………………………………………………...18
1. 細胞株…………………………………………………………………..18
2. 肺部初代幹細胞……………………………………….……….………19
(二) 實驗動物來源…………………………………………………………...19
(三) 試劑藥品………………………………………………………..….……19
(四) 儀器設備………………………………………………………………...20
第二節 實驗方法 …………………………………………………………….…22
(一) 紅色螢光奈米鑽石製備………………………………………………...22
(二) 細胞株實驗……………………………………………………………...23
1. 細胞株解凍………………………………………………..……………23
2. 細胞繼代培養 ……………………………………………….…………23
3. 螢光奈米鑽石標記3T3-L1及489-2.1細胞株……………..………….24
4. CFSE標記3T3-L1細胞株…………………………………………...…24
5. 流式細胞儀長時間追蹤標記的3T3-L1及489-2.1細胞株……………24
6. 細胞倍增時間分析……………………………………………………..25
7. 流式細胞儀分選螢光奈米鑽石標記的489-2.1細胞株………….……25
8. 細胞活性分析(Cell proliferation assay)………………………………..26
(三) 初代細胞實驗…………………………………………………………...26
1. 新生小鼠肺部幹細胞的萃取分離……………………………………..26
2. 肺部幹細胞的初代與繼代培養………………………………………..27
3. 螢光奈米鑽石標記肺部幹細胞與流式細胞分析技術………………..27
4. 活體內追蹤螢光奈米鑽石標記的肺部幹細胞………………………..28
5. 肺部組織切片製作……………………………………………………..29
6. 免疫組織化學染色(Immunohistochemistry, IHC)…………………….29
第四章 螢光奈米鑽石長時間標記與分選細胞株的研究………………………….32
第一節 實驗目的………………………………………………………………..32
第二節 實驗結果與討論………………………………………………………..32
第五章 螢光奈米鑽石活體內追蹤與分選初代肺部幹細胞的研究……………….47
第一節 實驗目的………………………………………………………………..47
第二節 實驗結果與討論………………………………………………………..48
第六章 結論………………………………………………………….………………52
參考文獻…………………………………66
附錄………………………………………………………….…………………………74

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陳奕穎，2010，〈螢光奈米鑽石及近紅外螢光染料之螢光能量共振轉移〉，國立台灣師範大學化學系碩士學位論文。

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