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研究生: 吳宇萱
Wu, Yu-Hsuan
論文名稱: 分子表面修飾的氧化鐵奈米粒子用於穩定人類降鈣素
Surface-decorated iron oxide nanoparticles used in stabilizing human calcitonin
指導教授: 杜玲嫻
Tu, Ling-Hsien
口試委員: 廖美儀
Liao, Mei-Yi
葉怡均
Yeh, Yi-Chun
杜玲嫻
Tu, Ling-Hsien
口試日期: 2022/06/27
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 64
中文關鍵詞: 人類降鈣素氧化鐵奈米粒子葉綠素金屬衍生物蘇木精
英文關鍵詞: human calcitonin, iron oxide nanoparticles, chlorophyll metal derivatives, hematoxylin
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202201014
論文種類: 學術論文
相關次數: 點閱:102下載:0
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  • 類澱粉蛋白纖維的形成在類澱粉蛋白變性中是重要的關鍵。此外,一些多肽類賀爾蒙,如人類降鈣素 (human calcitonin, hCT) ,因具有調節血鈣水平的作用而聞名,但由於hCT具高度傾向形成類澱粉蛋白纖維,因此作為藥物的潛力有限。
    奈米材料在各個科學領域具有許多優勢,通常具有獨特的物理及化學性質,如體積小、表面積大和與其他物質反應時活性極高。最近,研究指出氧化鐵奈米粒子經過適當表面修飾能夠抑制類澱粉蛋白纖維的形成。在這項研究中,我們發現通過不同方法合成的 Fe3O4@Chl/Fe (Fe-C) 、Fe3O4@Chl/Cu (Fe-CCu) 和 Fe3O4@hematoxylin (Fe-SU) 會影響hCT的成核和聚集過程。為了獲取雙重的驗證,我們使用兩種放光區域不同的苯並噻唑螢光探針來監測 hCT 類澱粉蛋白纖維的形成,以避免氧化鐵奈米粒子自身光學特性潛在的干擾,並使用電子顯微鏡觀察樣品終點樣貌。額外使用動態光散射粒徑分析儀輔助監測hCT纖維的形成 。由凝膠電泳來佐證出終點樣品的單體含量,和使用4,4'-二苯胺基-1,1'-聯二萘-5,5'-二磺酸與尼羅紅觀測共培育後的纖維含量。我們證實Fe-C為效果最佳的奈米粒子用作抑制hCT類澱粉蛋白纖維的產生,可使hCT更加穩定,經共培養後依然保留最多蛋白質單體,得以與細胞膜上的受體結合,增加其生物活性。此外,我們發現Fe-C以及Fe-CCu能夠降解預先形成的hCT纖維。我們的目標是希望在 hCT 製劑中添加一種新穎材料,以增加 hCT 作為藥物活性成分的機會。

    Accumulation of amyloid fibrils plays an important role in the development of amyloidosis. Besides, some hormone peptide such as human calcitonin (hCT) known for its effect in regulating blood calcium levels has limited pharmaceutical potential due to a high tendency to form amyloid.
    Nanotechnology offers many advantages in various fields of science, they usually have unique physicochemical properties such as small size, large surface area, and high reactivity. Recently, research has emphasized that appropriate surface modifications of nanoparticles can inhibit amyloid fibril formation. In this study, we found that Fe3O4@Chl/Fe (Fe-C), Fe3O4@Chl/Cu (Fe-CCu), and Fe3O4@hematoxylin (Fe-SU) synthesized via different approaches influence the nucleation and aggregation process of peptides. To obtain unbiased data, we used two different benzothiazole-based fluorescent probes to monitor hCT amyloid formation to avoid potential interference from the optical properties of nanoparticles. The appearance of hCT samples from the ThT endpoint was observed by transmission electron microscopy. Besides, the time course size distribution of hCT and nanoparticles mixture was monitored by dynamic light scattering. The monomer content from the ThT end product was checked by gel electrophoresis, and the fiber content of the ThT end product was measured using bis-ANS and nile red assay. The intracellular activated cAMP level was measured to confirm that Fe-C can inhibit hCT amyloid formation and major content with monomer type has larger remained. In addition, we found that Fe-C and Fe-CCu can dissociate pre-formed hCT fibrils. Our ultimate goal is to apply new material in hCT formulation to increase the opportunity of hCT to serve as active pharmaceutical ingredients in the medicine.

    謝誌 i 摘要 ii 目錄 iv 中英文對照表 vi 圖目錄 xiii 第一章 緒論 1 1.1 類澱粉蛋白病變 1 1.2 類澱粉蛋白結構與聚集機制 2 1.3 人類降鈣素 (human calcitonin, hCT) 4 1.4 抑制hCT纖維化之探討 7 1.5 氧化鐵奈米粒子及其表面修飾 11 1.6 氧化鐵奈米粒子應用於抑制類澱粉蛋白纖維形成之探討 13 1.7 研究目的及動機 15 第二章 實驗材料與方法 16 2.1 實驗材料 16 2.2 實驗方法 18 2.2.1 胜肽合成、純化及鑑定 18 2.2.2 氧化鐵奈米粒子的合成 22 2.2.3 樣品前處理及配製 23 2.2.4 動態光散射粒徑分析儀 (dynamic light scattering, DLS) 24 2.2.5 界面電位分析儀 (zeta potential) 26 2.2.6 吸收及螢光光譜 27 2.2.7 硫黃素T動力學測定 (thioflavin T assay, ThT assay) 28 2.2.8 穿透式電子顯微鏡 (transmission electron microscopy, TEM) 30 2.2.9 凝膠電泳 (gel electrophoresis) 31 2.2.10 等溫滴定量熱法 (isothermal titration calorimetry, ITC) 33 2.2.11 環腺苷酸活性測定 (cyclic AMP activity assay) 34 2.2.12 毒性測定 (MTT assay) 35 第三章 結果與討論 37 3.1 胜肽和合成與鑑定 37 3.2 蘇木精對hCT的抑制情形觀察 38 3.3 氧化鐵奈米粒子鑑定 39 3.4 氧化鐵奈米粒子對hCT的抑制行為 41 3.5 外力擾動條件下定時間監測氧化鐵奈米粒子對hCT聚集的影響 45 3.6 氧化鐵奈米粒子對hCT纖維的降解作用 53 3.7 降鈣素生物活性測試 55 第四章 結論 57 參考資料 58

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