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系統識別號 U0026-0309201513314000
論文名稱(中文) 雌激素鍵結之蛋白質金奈米團簇-合成與特徵分析及其應用
論文名稱(英文) Estrogen Functionalized Protein-Based Gold Nanoclusters - Synthesis, Characterization and Applications
校院名稱 成功大學
系所名稱(中) 化學系
系所名稱(英) Department of Chemistry
學年度 103
學期 2
出版年 104
研究生(中文) 蔡奇諺
研究生(英文) Chi-Yan Tsai
電子信箱 fantastic667@hotmail.com
學號 L36024074
學位類別 碩士
語文別 中文
論文頁數 85頁
口試委員 指導教授-陳淑慧
口試委員-李介仁
口試委員-徐睿良
中文關鍵字 金奈米團簇  雌激素  點擊化學  細胞顯影 
英文關鍵字 gold nanaclusters  estrogen  click chemistry  cell imaging 
學科別分類
中文摘要 以蛋白質所包覆合成的金奈米團簇(gold nanoclusters,NCs)具有好的螢光特性及生物相容性,蛋白質上多種胺基酸提供了可修飾不同分子的優點,使之成為具有功能性與專一性的探針。研究中在牛血清蛋白質金奈米團(BSA-AuNCs)表面修飾上雌激素(17α-ethinyl estradiol, EE2),利用一段聚乙二醇(polyethylene glycol, PEG)具有疊氮基(azide)和胺基(amine),雌激素上的炔基(alkyne)能與疊氮基經由銅金屬催化行環化加成反應,形成1,4-雙取代-1,2,3-三氮唑的五員雜環。而PEG另一端胺基則可利用交聯試劑與蛋白質上的羧酸基經由醯胺化反應鍵結。將此合成出來的E2-BSA-AuNCs應用在乳癌細胞顯影上,在具有雌激素受體的MCF-7乳癌細胞中,E2-BSA-AuNCs會隨著時間由細胞膜慢慢進入細胞核,在時間2小時時已完全進入細胞核,推測E2-BSA-AuNCs是經由細胞膜的受體辨認而進入到細胞中並進入到細胞核中,而對照組BSA-AuNCs則無法進入細胞內;另以市售螢光染料Cyanine 3上修飾上雌激素(E2-Cy3)作為比較,反應時間在6小時才進入到細胞核,表示E2-BSA-AuNCs具有更好的生物相容性。而在不具雌激素受體MD-MBA-231乳癌細胞實驗結果顯示,E2-BSA-AuNCs不具有螢光訊號表現,但是E2-Cy3在細胞質卻有螢光表現,推測E2-Cy3是藉由被動傳輸進入到細胞中。另外以MTT試驗測試比較細胞增殖速率,實驗結果顯示在相同的藥品濃度下E2-BSA-AuNCs比E2-PEG具有較高的增殖率,顯示所合成的E2-BSA-AuNCs更容易進入到細胞核中作用。實驗中所合成的BSA-AuNCs及E2-BSA-AuNCs以質譜儀作為鑑定,金奈米團簇為15個金原子所組成,而有5個E2-PEG鍵結在蛋白質表面;而在原子力顯微鏡下,有修飾E2-PEG的粒子尺寸比未修飾的直徑變大約2nm;而E2-PEG與E2-Cy3的合成鑑定,經由質譜儀與核磁共振光譜作為鑑定。
英文摘要 Protein-protected fluorescent gold nanoclusters (AuNCs) have attracted many attentions due to their superior properties such as low toxicity, high biocompatibility and feasible functionalization. In this study, bovine serum albumin-protected AuNCs (BSA-AuNCs) was synthesized and functionalized with 17β-estradiol-azide-poly(ethylene glycol) conjugates (E2-PEG) via carbodiimide crosslinker chemistry (EDC/NHS) and used to selectively target estrogen receptor positive breast cancer cell. E2-PEG derivative was synthesized from Ethinyl Estradiol (EE2) by CuI-assisted azide-alkyne cycloaddition. The products, BSA-AuNCs and E2-BSA-AuNCs were purified by gel filtration chromatography or sucrose density gradient and characterized by fluorescence spectroscopy. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Fourier Transform Infrared spectroscopy (FT-IR), Matrix assisted laser desorption mass spectroscopy (MALDI-MS). The data showed each BSA-AuNCs contains eighteen Au atoms and five E2-PEG molecules with an emission maximum at around 640 nm (380 nm excitation) which was not much altered by E2-PEG conjugation. Furthermore, the volume of BSA-AuNCs (or E2-BSA-AuNCs) was found to expand by four times as compared to that of the native BSA. This resulted in a 4-fold decreased in density as observed by sucrose density gradient and yielded a loosely packed E2-BSA-AuNCs assembly. Based on a time course study using confocal fluorescence microscopy, E2-BSA-AuNCs were shown to selectively target estrogen receptor positive MCF-7(ER+) breast cancer cell by membrane ER assisted endocytosis rather than by passive diffusion of the free drug. In contrast, E2-Cy3 could not differentiate MCF-7(ER+) versus MDA-MB-231(ER-) cell due to the passive transport of the free estrogen. MTT assay reveals that AuNCs conjugation increases the estrogen potency by 1.6 folds due to increased rates of nuclei transport.
論文目次 Abstract I
中文摘要 III
誌謝 X
目錄 XI
圖目錄 XIV
表目錄 XVI
簡稱用語對應表 XVII
第一章 研究內容 1
1.1研究動機 1
1.2研究策略與方向 2
第二章 文獻回顧 3
2.1金奈米團簇(Gold Nanoclusters)簡介 3
2.1.1不同模板的金奈米團簇 3
2.1.2金奈米團簇發光原理 13
2.1.3蛋白質金奈米團簇在生物顯影上的應用 14
2.2雌激素簡介 18
2.2.1雌激素與乳癌相關性 18
2.2.2雌激素與雌激素受體作用途徑機制 19
第三章 實驗部分 23
3.1實驗藥品與儀器 23
3.1.1實驗藥品 23
3.1.2實驗儀器 24
3.2 牛血清蛋白包覆金奈米團簇(BSA-AuNCs) 24
3.2.1 BSA-AuNCs合成 24
3.2.2 BSA-AuNCs純化 25
3.3 雌激素與N3-PEG-NH2鍵結 26
3.3.1 E2-PEG合成 27
3.3.2 E2-PEG純化 28
3.4 E2-PEG-NH2與BSA-AuNCs的醯胺化反應 29
3.5 E2與Cyanine 3染料的鍵結(E2-Cy3)合成與純化 30
3.6 乳癌細胞分析實驗 32
3.6.1 細胞培養 32
3.6.2 MTT細胞分析實驗 33
3.6.3 細胞顯影 33
3.6.4 雌激素競爭實驗 34
第四章 實驗結果與討論 35
4.1 BSA-AuNCs 35
4.1.1光學特性 35
4.1.2金奈米團簇分析 36
4.1.3溶劑穩定性分析 37
4.1.4樣品純化與質譜分析 38
4.2 E2-PEG 40
質譜儀鑑定E2-PEG 41
1H NMR鑑定E2-PEG 41
4.3 E2-BSA-AuNCs 41
4.3.1光學特性 41
4.3.2傅立葉轉換紅外線光譜分析(Fourier transform infrared spectroscopy,FTIR) 42
4.3.3原子力顯微鏡分析(Atomic force microscopy, AFM) 43
4.3.4質譜分析 43
4.4 E2-Cy3純化與鑑定 44
4.5細胞實驗結果 44
4.5.1 細胞顯影 44
4.5.2 MTT分析實驗 46
4.5.3 雌激素競爭實驗 47
第五章結論 77
第六章參考文獻 78
附錄一 82
附錄二 83
附錄三 84
附錄四 85
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