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研究生: 黃景鴻
Huang, Ching-Hong
論文名稱: 以材料為核心探索 : 透過抑制聚集以穩定人類降鈣素
A material-centric exploration : Stabilizing human calcitonin by inhibiting aggregation
指導教授: 杜玲嫻
Tu, Ling-Hsien
口試委員: 杜玲嫻
Tu, Ling-Hsien
李以仁
Lee, I-Ren
葉伊純
Yeh, Yi-Cheun
口試日期: 2024/07/30
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2024
畢業學年度: 112
語文別: 中文
論文頁數: 73
中文關鍵詞: 人類降鈣素類澱粉蛋白聚集史托伯法
英文關鍵詞: human calcitonin, amyloid aggregation, Stöber process
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202401606
論文種類: 學術論文
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  • 人類降鈣素(human calcitonin, hCT)是一種由32個胺基酸所組成的荷爾蒙胜肽。在生物體內扮演調節血鈣水平的重要角色,可抑制破骨細胞的活動,因此常被作為藥物使用來治療骨骼相關疾病,例如: 佩吉特氏病和骨質疏鬆症等。由於它高聚集傾向的特性,導致其藥物效果受到一定限制,我們的研究目標在於開發新的材料用於穩定該胜肽。根據先前實驗室的研究發現,醣類分子對抑制hCT的聚集具有潛在能力,但需要在高濃度下才能發揮聚集抑制的作用。在這項研究中,我們利用史托伯法 (Stöber process )製備溶膠,並引入額外的葡萄糖,希望能有效地串聯溶液中的葡萄糖分子,藉此降低葡萄糖的濃度或提升抑制聚集的效果。在本研究中,我們參考了相關文獻中的製備條件,以獲得穩定的樣品溶液,之後再進行製備條件的優化。當製備出的材料穩定度提升後,我們才開始調整添加的葡萄糖濃度。首先,透過凝膠滲透色譜(Gel permeation chromatography, GPC)判斷材料的交聯程度,確保其交聯程度相似後,再以傅立葉轉換紅外光譜(Fourier-transform infrared spectroscopy, FTIR)確認葡萄糖是否交聯於材料中。接著,我們利用X射線光電子能譜(X-ray photoelectron spectroscopy, XPS)分析材料之元素比例 ,推算出材料中交聯的葡萄糖量。在硫磺素T(Thioflavin T, ThT)動力學實驗中,我們觀察到材料通過減少共同培育下的hCT纖維數量方式達到抑制hCT聚集的效果,該結果也透過穿透式電子顯微鏡(Transmission electron microscopy, TEM)得到驗證。

    Human calcitonin (hCT) is a hormonal peptide composed of 32 amino acids. It plays a crucial role in regulating blood calcium levels in the body by inhibiting the activity of osteoclasts. Therefore, it is often used as a medication to treat bone-related diseases, such as Paget's disease and osteoporosis. Due to its high tendency to aggregate, the efficacy of hCT as a drug is somewhat limited. Our research aims to develop new materials to stabilize this peptide. Previous studies in our laboratory have found that carbohydrate molecules have the potential to inhibit the aggregation of hCT, but they require high concentrations to be effective. In this study, we use the Stöber process to prepare sols and introduce additional glucose, hoping to effectively link the glucose molecules in the solution, thereby reducing the glucose concentration or enhancing the aggregation inhibition effect. In this study, we referred to the preparation conditions in the relevant literature to obtain a stable sample solution, and then optimized the preparation conditions later. Once the stability of the prepared material was improved, we started to adjust the concentration of added glucose. First, we determined the degree of crosslinking of the materials using gel permeation chromatography (GPC) to ensure similar crosslinking levels. We then used Fourier-transform infrared spectroscopy (FTIR) to confirm whether glucose was crosslinked within the materials. Next, we utilized X-ray photoelectron spectroscopy (XPS) to analyze the elemental composition of the materials and estimate the amount of glucose crosslinked within them. In thioflavin T (ThT) kinetic experiments, we observed that the materials inhibited hCT aggregation by reducing the number of hCT fibers when co-incubated. This result was further verified by transmission electron microscopy (TEM).

    第一章 緒論 1 1.1 類澱粉蛋白與相關病變 1 1.2 類澱粉蛋白的聚集機制 3 1.3 類澱粉蛋白聚集體偵測 4 1.4 降鈣素與其在生理上的功能 6 1.5 人類降鈣素聚集造成的影響與聚集機制探討 8 第二章 研究核心 11 2.1 人類降鈣素抑制文獻探討 11 2.2 滲透物及其作為hCT聚集抑制劑的潛力 13 2.3 以材料串聯功能性分子的潛力 15 2.4 研究動機 17 第三章 實驗材料與方法 18 3.1 實驗材料與儀器設備 18 3.2 胜肽合成與純化鑑定 21 3.2.1 胜肽合成 21 3.2.2 胜肽純化與鑑定 24 3.3 胜肽樣品前處理以及配製 27 3.4 硫磺素T動力學測定 (Thioflavin-T kinetic assay) 28 3.5 穿透式電子顯微鏡 (Transmission electron microscopy, TEM) 29 3.6 材料製備 30 3.7 傅立葉轉換紅外線光譜法 (Fourier-transform infrared spectroscopy, FTIR) 32 3.8 動態光散射 (Dynamic light scattering, DLS) 34 3.9 X射線光電子能譜學 (X-ray photoelectron spectroscopy, XPS) 36 3.10 凝膠滲透色譜法 (Gel permeation chromatography, GPC) 37 第四章 結果與討論 39 4.1 材料製備的優化 39 4.1.1 交聯時間與鹼催化劑濃度的影響 39 4.1.2 反應溫度調整與透析處理 41 4.2 材料交聯程度鑑定 43 4.3 葡萄糖參與交聯反應的證據 44 4.4 材料元素組成分析 46 4.5 探討材料對於類澱粉蛋白聚集抑制的影響 47 4.5.1 胜肽合成鑑定 47 4.5.2 ThT測定材料影響hCT聚集動力學 49 4.6 其他條件材料探討 53 第五章 結論 58 參考資料 59 附錄 65

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