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研究生: 陳育材
Yu-Cai Chen
論文名稱: 應用特殊修飾金奈米粒子與金屬離子控制類澱粉樣纖維之生長
Control of Amyloid Aggregation by Applying Gold Nanoparticles and Metal Ions
指導教授: 黃人則
Jen-Tse Huang
何明樺
Ming-Hua Ho
口試委員: 郭俊宏
Chun-Hong Kuo
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 131
中文關鍵詞: 奈米粒子金屬離子神經退化性疾病阿茲海默症肌萎縮性脊隨側索硬化症亨丁頓氏舞蹈症
外文關鍵詞: metal ions, Amyotrophic Lateral Sclerosis
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  • 本研究是利用金奈米粒子與不同之金屬離子去調控神經退化性疾病(亨丁頓氏舞蹈症與肌萎縮性脊髓側索硬化症)之類澱粉樣纖維化聚集結構的生長,本論文分為兩個部分,第一部分為將金奈米粒子應用於亨丁頓氏舞蹈症,並探討其相關結果;而第二部分為探究肌萎縮性脊髓側索硬化症中之關鍵序列(TDP-43)與不同金屬離子之間的交互作用力。其詳細結果如下:
    第一部分為利用特殊設計之胜肽鏈搭載金奈米粒子,應用於亨丁頓氏舞蹈症之相關研究,並以KKQ20K之類澱粉樣纖維化樣版模型,藉由紫外可見光光譜儀、X光射線光電子能譜儀與穿隧式電子顯微鏡等儀器進行後續的分析與鑑定。結果顯示,特殊修飾後之金奈米粒子具有抑制KKQ20K之纖維化聚集結構的生長,並於穿隧式電子顯微鏡影像圖中發現,具有摧毀KKQ20K之纖維結構之能力;在與不同之神經退化性疾病之共培養下,顯示出特殊修飾之金奈米粒子對富含麩醯胺酸之胜肽鏈具有明顯的親和力。另一方面,在細胞實驗中,我們選用了大量表達具有eYFP標記之聚麩醯胺酸蛋白質之細胞進行實驗,為了增加細胞之包吞作用,我們利用富含正電性之聚乙烯亞胺進行修飾,在顯微鏡結果中顯示出利用特殊修飾之金奈米粒子,大幅度且清楚的分散了聚麩醯胺酸之聚集體。從細胞內與細胞外之實驗顯示出特殊修飾之金奈米粒子具有親和性的辨識聚麩醯胺酸並且加以抑制、摧毀其纖維化聚集結構之能力。
    第二部分為利用不同之金屬離子(鋅、鋁、銅)與肌萎縮性脊髓側索硬化症之關鍵序列(D1與QN1)共培養的情況下,並且利用圓二色光譜儀、螢光光譜儀等儀器進行觀測及分析。結果顯示,對QN1而言,鋅離子會造成更多更明顯得-sheet纖維狀聚集結構的生成,反之,銅離子則會形成不規則的聚集型態,而鋁離子則無顯著的影響;對D1來說,銅離子會加速纖維狀結構的聚集,而鋁離子則使之結構維持在Random coil的形式。造成如此大的差異性存在,可能來自胜肽鏈序列的不同,使得推疊結構不同與金屬之間的交互作用力亦不同,在接下來的實驗中,希望可以利用更進一步的分析方法加以驗證。


    In our study, we use the gold nanoparticles and different metal ions to control the formation of amyloid fiber structure of the neurodegenerative diseases (like as huntingtin’s disease (HD) and amyotrophic lateral sclerosis (ALS)).
    There are two part in my dissertation, part A is based on the rational designed amyloid-sensitive gold nanoparticles (AuNPs) to apply in huntingtin’s disease and we use the KKQ20K as the model study of huntingtin’s disease. By the result of the UV/Vis spectroscopy, the X-ray photoelectron spectroscopy (XPS) and the transmission electron microscopy (TEM), indicating the AuNPs can prevent the formation of KKQ20K amyloid aggregation, and the TEM image also showing that AuNPs have the ability to destroy the fiber form of KKQ20K. On the other hand, we co-incubated the AuNPs with another peptide of the neurodegenerative diseases demonstrate that the AuNPs can affinity to interact with the polyglutamine (polyQ)-rich peptide. In cell study, we choose the eYFP-tagged mutant Huntingtin (109Q) protein to do the experiment. In order to increase the uptake of cell, we coating a polyethyleneimine (PEI) layer on the surface of AuNPs. From the cell study, showing that the AuNPs can dissociate the large cytosolic aggregates.
    In the part B, we use the different metal ions co-incubation with D1 and QN1 of the c-terminal in TAR DNA-binding protein 43 of ALS. In the result of the circular dichroism spectrum and the fluorescence spectrometry show that, for QN1, zinc ion cause more and stronger β-sheet structure, copper ion result in forming amorphous aggregates, and the influence of aluminum ion was slightly. For D1, copper ion will accelerate the aggregation, and conversely aluminum ion cause D1 maintain random coil signal of CD spectrum. The huge difference between the QN1 and D1 affected metal ions, maybe is due to the sequence causing the stacking structure and the interaction with metal ions are very different. This hypothesis will be prove by further analysis.

    中文摘要 I 英文摘要 III 目錄 V 圖目錄 VIII 表目錄 XII 第一章 緒論 1 1.1 神經退化性疾病 1 1.1.1 多麩醯胺酸神經退化性疾病(Polyglutamine Disease) 2 1.1.1.1 多麩醯胺酸神經退化性疾病:亨丁頓氏舞蹈症(Huntington's Disease, HD) 4 1.1.1.2 亨丁頓氏蛋白質 7 1.1.2 肌萎縮性脊髓側索硬化症(Amyotrophic lateral sclerosis, ALS) 9 1.1.2.1 TAR DNA-binding protein43 12 1.1.3 阿茲海默症(Alzherimer's Disease, AD) 13 1.1.3.1 Amyloid beta(Aβ)與Tau protein 16 1.1.4 蛋白質高度聚集化與神經退化性疾病的關聯性 19 1.2 奈米粒子 20 1.2.1 奈米粒子的種類 22 1.2.2 奈米粒子的表面修飾與鑑定 27 1.2.2.1 奈米粒子的表面修飾技術(分子自組裝) 27 1.2.2.2 自組裝分子的種類 29 1.2.2.3 表面修飾之特性鑑定 32 1.3 金屬離子與神經退化性疾病之間的交互作用力 34 1.4 胜肽合成 36 1.4.1 液相胜肽合成 37 1.4.2 固相胜肽合成 38 1.4.3 微波輔助固相胜肽合成 43 第二章 實驗器材與方法介紹 48 2.1 實驗設計與原理 48 2.1.1 利用特殊修飾之金奈米粒子應用於亨丁頓氏舞蹈症 48 2.1.2 探討金屬離子與神經退化性疾病之交互作用力 52   2.2 實驗儀器與藥品 53 2.2.1 實驗儀器 53 2.2.2 實驗藥品 54 2.3 微波輔助固相胜肽合成儀(Microwave Assisted Solid Phase Peptide Synthesizer) 55   2.4 螢光分子基團的修飾 57 2.5 奈米粒子 58 2.6 奈米粒子的表面修飾 60 2.7 高效液相層析(High Performance Liquid Chromatography, HPLC) 62 2.8 紫外可見光光譜儀(UV/Vis Spectroscopy) 65 2.9 質譜儀(Mass Spectrometer) 66 2.10 動態光散射粒徑分析儀及界面電位分析儀(Particle Size and Zeta Potential Analyzer) 69 2.11 圓二色光譜儀(Circular Dichroism, CD) 71 2.12 酶聯免疫吸附試驗(Enzyme-Linked Immunosorbent Assay, ELISA) 73 2.13 穿隧式電子顯微鏡(Transmission Electron Microscopy, TEM) 75 2.14 X光射線光電子能譜儀(X-ray Photoelectron Spectroscopy, XPS) 77 2.15 多麩醯胺酸序列聚集化之抑制與摧毀 79 2.16 Probe的專一性測試 81 2.17 細胞樣品的培養與製備 82 第三章 實驗結果與討論(Part A) 83   3.1 胜肽鏈之性質鑑定 83 3.2 奈米粒子的特性鑑定 86 3.2.1 金奈米粒子的特性鑑定 86 3.2.2 官能化後的金奈米粒子之特性鑑定 88 3.3 KKQ20K之抑制纖維化聚集結構測試 93 3.4 KKQ20K之摧毀纖維化聚集結構測試 97 3.5 JLD2之專一性測試暨修飾後金奈米粒子與KKQ20K之交互作用力 102 3.6 細胞實驗 106 第四章 實驗結果與討論(Part B) 108 4.1 胜肽鏈之性質鑑定 108 4.2 胜肽鏈與金屬離子間之交互作用力 110 第五章 結論 118 第六章 參考文獻 123

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