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研究生:胡志達
研究生(外文):Hu Chih Ta
論文名稱:以KKQ40KK當作杭丁頓舞蹈症的胜肽模型做其相關性質的研究
論文名稱(外文):KKQ40KK As a Model Peptide for Huntington's Disease Related Studies
指導教授:黃人則程中玉
指導教授(外文):Huang, Joseph Jen-TseCherng, Jong-Yuh
口試委員:黃人則程中玉李政怡
口試委員(外文):Huang, Joseph Jen-TseCherng, Jong-YuhLee, Cheng-I
口試日期:2014-07-22
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學暨生物化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:66
中文關鍵詞:杭丁頓舞蹈症神經退化性疾病
外文關鍵詞:Huntington's DiseaseKKQ40KK
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杭丁頓舞蹈症 (Huntington's disease HD) 是一種神經退化性疾病,造成其疾病原因是染色體異常所導致的疾病,杭丁頓蛋白基因片段中CAG的異常重複將會轉錄再轉譯為過長的聚麩醯胺酸(polyQ),過長的聚麩醯胺酸可能會造成錯誤的蛋白質摺疊,聚集成不能被溶解的聚集物,造成其神經元的凋亡。
KKQ40KK模擬杭丁頓蛋白中聚麩醯胺酸片段。為了觀察TDP-43蛋白中C端與杭丁頓蛋白中聚麩醯胺酸的作用,我們以不同的晶種(D1、QN1)加入聚麩醯胺酸胜肽KKQ40KK中並觀察聚集化的影響。
而實驗室開發的胜肽藥物Compound X,其在細胞與老鼠實驗中已被證實是具有抑制杭丁頓蛋白聚集的能力。我們觀察將Compound X加入到KKQ40KK。以CD去觀察二級結構的變化,ThT做其類澱粉纖維程度的比較,以及TEM做類澱粉纖維的影像觀察。
在引晶的實驗中,我們成功的觀察到了加入晶種影響聚麩醯胺酸的聚集的現象。而在加入Compound X後的KKQ40KK二級結構觀察,我們實驗結果發現KKQ40KK有更傾向β-sheet結構的形成。
我們推測富含Q/N的胜肽QN1較D1能影響杭丁頓蛋白的聚集。並且,Compound X的作用在這個胜肽模型中與細胞實驗結果不符,指出此系統並不能完全代表杭丁頓氏舞蹈症的不正常蛋白聚集。

The neurodegenerative disorder Huntington's disease (HD) is caused by a mutation in a curious protein called huntingtin. Unlike many other proteins of a similar size (348 kDa), huntingtin is completely soluble.
The disease-causing gene mutation consists of an expanded CAG tract (>35 repeats) at the 5′ end, which is translated into a corresponding polyglutamine stretch (polyQ) that makes medium spiny neurons in the striatum particularly vulnerable to cell death, and also leads to the dysfunction and death of neurons in other brain regions.
We use polyQ peptide KKQ40KK as a huntingtin model to study the interaction with other biomolecules. By using different peptide fiber (D1Core and QN1) as a seed toward polyQ peptide in order to observe the interaction of TDP-43 protein C-terminus and polyQ.
By adding compound X to peptide KKQ40KK, we observe the secondary structure transformation by Circular Dichroism (CD), amyloid fiber formation by Thioflavin T (ThT) fluorescence emission, and fiber morphology change by transmission electronic microscopy (TEM).
We successfully observe that the seeds can affect the polyglutamine aggregation. In addition, after Compound X is added to the KKQ40KK, we find out that KKQ40KK can form -sheet structure more easily. This result is different to what we expect that the -sheet structure should reduce. This result might related to the buffer condition or the peptide concentration.
We speculate that the Q/N rich peptide could affect the aggregation of Huntintin protein more than D1. Moreover, the effect of Compund X is different with the cell experiments, which might imply that this system is not completely suitable to the abnormal protein aggregation in Hintinton’s Disease.


目錄:
誌謝
中文摘要
Abstract
第一章、緒論
1.1神經退化性疾病
1.1.1蛋白質錯誤折疊
1.2杭丁頓氏舞蹈症
1.2.1杭丁頓蛋白
1.2.2杭丁頓蛋白質的高度聚集化和神經退化性疾病
1.3肌萎縮性脊髓側索硬化症
1.3.1肌萎縮性脊髓側索硬化症
1.3.2 TAR DNA-binding protein 43
1.4研究目的與實驗流程
1.4.1研究目的
1.4.2實驗流程
第二章、實驗器材與方法
2.1實驗儀器與藥品
2.1.1實驗儀器
2.1.2實驗藥品
2.2固相胜肽合成法
2.3高效能液相層析儀
2.4螢光光譜儀
2.5圓二色光譜儀
2.6穿透式電子顯微鏡
2.7西方點墨法
第三章、實驗結果與討論
3.1聚麩醯胺酸胜肽與胜肽藥物
3.1.1聚麩醯胺酸胜肽溶解度實驗
3.2聚麩醯胺酸胜肽與TDP-43類澱粉纖維引晶效應實驗
3.3聚麩醯胺酸胜肽與胜肽藥物的結構與性質
3.3.1聚麩醯胺酸胜肽與胜肽藥物的硫磺素T螢光吸收光譜
3.3.2聚麩醯胺酸胜肽與胜肽藥物的二級結構變化
3.3.3聚麩醯胺酸胜肽與胜肽藥物的微影像
3.4胜肽藥物的改良
第四章、實驗總結
參考資料


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