臺灣博碩士論文加值系統

理想的細胞追蹤試劑需具備高生物相容性、易於使用、抗光漂白和長時間標定。奈米碳點（Carbon dots, CDs）具有高水溶性、低毒性、光穩定性和螢光可調性，適合作為細胞追蹤試劑應用在生物感測及生物影像等方面。本研究透過微波裂解法利用低分子量與高分子量聚乙烯亞胺（Polyethylenimine, PEI）合成 CD-Ls 及 CD-Hs 兩種 CDs ，並將之與透明質酸（Hyaluronic acid, HA）接枝，形成 CD-L-HAs 及CD-H-HAs。CDs 均可發出藍色螢光，其粒徑小於 10 nm 且表面電位為正。在細胞實驗部分，結果證明 CD-Ls 與 CD-L-HAs 對人類骨髓間葉幹細胞（marrow mesenchymal stem cells, MSCs）無細胞毒性，但 CD-Hs 在 30 μg/ml 與CD-H-HAs 在 50 μg/ml 有明顯細胞毒性；做為負向控制組的小鼠纖維母細胞（NIH3T3）分別與CD-L-HAs及 CD-H-HAs 共培養發現無細胞毒性。細胞攝入量部分，在 MSCs 能觀察到藍色螢光，而 NIH3T3 細胞並無發現螢光。細胞攝入螢光量在 MSCs 與 NIH3T3 間無顯著差異。活體螢光分布結果發現 0 小時能觀察到 CDs 的螢光表現，24小時候在 CD-Ls 組別仍能看見螢光。本研究成功開發出將 CDs 作為追蹤 MSCs 的成像劑之可能性。

Cell tracking labels with excellent biocompatibility, ease of use, resistance to photobleach and long-lasting efficiency are needed. Carbon nanodots (CDs) have high water solubility, low toxicity, photostability and fluorescent property which can be used in the areas of biosensing, bioimaging, light emitting device and drug carrier. In this study, carbon dots low molecules (CD-Ls) and carbon dots high molecules (CD-Hs) were synthesized by low molecular weight and high molecular weight polyethyleneimine (PEI) by microwave assisted pyrolysis. Furthermore, CD-Ls and CD-Hs were conjugated with hyaluronic acid (HA) form CD-L-HAs and CD-H-HAs. CDs were discovered they can emit blue fluorescence by UV light excitation, the particle size less than 10 nm according Atomic force microscopy and had positive zeta potential. In vitro assay revealed that CD-Ls and CD-L-HAs had no cytotoxicity against human bone marrow mesenchymal stem cells (MSCs). But 30 μg/ml of CD-Hs and 50 μg/ml of CD-H-HAs had significant cytotoxicity against MSCs. NIH3T3 cells which is negative control of MSCs cultured with CD-L-HAs or CD-H-HAs respectively had high cell viability. In cell uptake assay that MSCs had blue fluorescence. But NIH3T3 cells had not any fluorescence. In cell uptake of CDs’s fluorescence had no significant difference. The results of non invasion in vivo imaging system (IVIS) showed CDs’s fluorescence was observed at 0 hours. And CD-Ls still had blue fluorescence after 24 hours. Here, we provide the possibility of applying CDs as a label to tracking MSCs migration after systemic infusion into animals.

目錄
摘要 I
Abstract II
目錄 III
圖索引 VII
表索引 IX
縮寫表 X
第一章 緒論 1
1.1 前言 1
1.2 文獻回顧 3
1.2.1 奈米碳點的合成方法及物理化學特性 3
1.2.2 透明質酸標靶骨髓間葉幹細胞 5
1.3 研究動機與目的 8
第二章 材料與方法 9
2.1 奈米碳點製備 9
2.2 透明質酸與奈米碳點之改質接枝 10
2.2.1. 透明質酸接枝低分子量奈米碳點製備實驗流程 11
2.2.2. 透明質酸接枝高分子量奈米碳點製備實驗流程 12
2.3 紫外光/可見光光譜儀分析 12
2.4 螢光光譜儀分析 13
2.5 量子產率分析 13
2.6 核磁共振儀分析 14
2.7 傅立葉轉換紅外線光譜儀分析 14
2.8 跑膠電性測定 15
2.9 表面電位量測 16
2.10 質譜儀分析 16
2.11 原子力顯微鏡觀察 16
2.12 穿透式電子顯微鏡觀察 18
2.13 細胞培養 18
2.13.1. 人類不朽化骨髓間葉幹細胞 18
2.13.2 小鼠纖維母細胞培養基 20
2.14 細胞無菌培養 22
2.14.1 細胞繼代 22
2.14.2 細胞計數 23
2.14.3 細胞凍存 23
2.14.4 細胞解凍 24
2.15 細胞存活率分析 24
2.16 細胞攝入量分析 26
2.17 細胞攝入奈米碳點之螢光量分析 27
2.18 活體影像系統分析 27
2.19 統計分析 28
第三章 結果 29
3.1 奈米碳點合成與特性分析 29
3.1.1 紫外光/可見光光譜儀分析 30
3.1.2 螢光光譜儀分析 31
3.1.3 量子產率分析 33
3.1.4 核磁共振儀官能基分析 34
3.1.5 傅立葉轉換紅外線光譜儀分析 36
3.1.6 跑膠電性測定 39
3.1.7 表面電位量測 40
3.1.7 質譜儀之分子量分析 41
3.1.8 原子力顯微鏡觀察 43
3.1.9 穿透式電子顯微鏡觀察 44
3.2 奈米碳點對細胞之生物相容性 45
3.2.1 奈米碳點對人類不朽化骨髓間葉幹細胞之生物相容性評估 45
3.2.2 奈米碳點對小鼠纖維母細胞之生物相容性評估 47
3.3 細胞攝入奈米碳點之螢光影像 49
3.3.1 人類不朽化骨髓間葉幹細胞攝入奈米碳點之螢光影像 49
3.3.2 小鼠纖維母細胞攝入奈米碳點之螢光影像 54
3.4 細胞攝入螢光量分析 57
3.5 奈米碳點之小鼠螢光分布 58
第四章 討論 61
4.1 低分子量奈米碳點與高分子量之特性比較 61
4.2 奈米碳點對細胞之生物相容性 62
4.3 奈米碳點之細胞攝入量與螢光量之比較 62
4.4 奈米碳點之活體螢光分布 63
第五章 結論 64
參考文獻 65
附錄 73
A. 儀器表 73
B. 藥品表 75

圖索引
圖 1-1 研究架構 .................. 8
圖 2-1 透明質酸結構圖 ............ 10
圖 2-2 EDC/NHS 交聯原理 .......... 11
圖 2-3 人類不朽化骨髓間葉幹細胞型態...... 19
圖 2-4 小鼠纖維母細胞型態 .................... 21
圖 3-1 奈米碳點溶液被白光及紫外光激發 ............. 29
圖 3-2 奈米碳點之紫外光/可見光吸收光譜 ............. 30
圖 3-3 奈米碳點吸光/螢光光譜 .................. 32
圖 3-4 CDs 之量子產率 ....................... 33
圖 3-5 1H-NMR 圖譜 ..................... 35
圖 3-6 CDs 之 FTIR 圖譜 ....................... 38
圖 3-7 CDs 之跑膠電性測定 ....................... 39
圖 3-8 CDs 之表面電位 .................. 40
圖 3-9 CD-Ls、CD-L-HAs 和 HA 之質譜儀分析 ............. 42
圖 3-10 奈米碳點之 AFM 影像 ..................... 43
圖 3-11 奈米碳點之 TEM 影像 ................... 44
圖 3-12 CDs 與 MSCs 之細胞存活率 .............. 46
圖 3-13 CDs 與 NIH3T3 細胞之細胞存活率 ................ 48
圖 3-14 螢光顯微鏡觀察 MSCs 攝入 CD-Ls 之影像 .......... 50
圖 3-15 螢光顯微鏡觀察 MSCs 攝入 CD-Hs 之影像 .......... 51
圖 3-16 螢光顯微鏡觀察 MSCs 攝入 CD-L-HAs 之影像 ....... 52
圖 3-17 螢光顯微鏡觀察 MSCs 攝入 CD-H-HAs 之影像 ....... 53
圖 3-18 螢光顯微鏡觀察 NIH3T3 細胞攝入 CD-L-HAs 之影像 . 55
圖 3-19 螢光顯微鏡觀察 NIH3T3 細胞攝入 CD-H-HAs 之影像 . 56
圖 3-20 NIH3T3 細胞及 MSCs 攝入螢光比較................ 57
圖 3-21 未注射奈米碳點前之 IVIS 螢光比較................ 59
圖 3-22 注射奈米碳點後之 IVIS 螢光結果 ................. 59
圖 3-23 IVIS 之 ROI 柱狀圖 ................ 60

表索引
表 2-1 細胞存活率分析之奈米碳點溶液濃度 ............... 25
表 2-2 細胞攝入量分析之奈米碳點溶液濃度 ............... 26
表 2-3 細胞攝入量分析之奈米碳點溶液濃度 ............... 27
表 3-1 CDs 之 FTIR 特徵吸收峰比較 ............... 37

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