中文摘要
阿茲海默症是神經退化性疾病的一種主要疾病，其主要的病理特徵是神經細胞內纖維化糾結以及腦細胞外老年斑塊，而貝它類澱粉蛋白的聚集則是造成老年斑塊的主要原因。儘管貝它類澱粉蛋白已經被廣泛的研究，但其沉積聚集的機制尚未清楚。本次實驗旨在探討膠原蛋白序列對貝它類澱粉蛋白的抑制效果，並分析貝它類澱粉片段序列蛋白對膠原蛋白三股螺旋結構穩定性、摺疊速率的影響。
本研究中，我們選擇貝它類澱粉蛋白中最重要的一段胺基酸序列 A(16-12) ( KLVFFAE) 來做研究，合成一系列接上膠原蛋白相關序列 (POG)n 之 A 相關胜肽鏈共八段: 未接上膠原蛋白序列 (WT-A (16-22))，在碳端的位置接上六個POG( A(POG)6 )、七個 POG ( A(POG)7 ) 、十個 POG ( A(POG)10 )，在氮端位置接上七個 POG ( (POG)7A ) 、十個 POG ( (POG)10A ) 及單純的膠原蛋白 (POG)7、(POG)10。利用螢光光譜儀、TEM、CD、DLS以及 FT-IR來探討這些胜肽鏈是否對 A 沉積具有抑制的效果。
經由 CD 測量發現 A(16-22) 位於胜肽鏈之 N 端時會妨礙膠原蛋白三股螺旋結構的摺疊，使熱穩定性降低；但 A(16-22) 位於胜肽鏈之 C 端時則會幫助三股螺旋結構的摺疊使熱穩定性上升。從觀察 A 聚集現象我們發現無論在 A (16-22) 的 C 端或 N 端接上七個 POG 都不會有聚集現象的產生，但接上十個 POG 後反而會加強聚集現象，因此我們知道 POG 的數目不宜過多才會有較好的抑制效果，最後將 POG 數目較少的三條胜肽: A(POG)6、 A(POG)7、(POG)7A 與 WT-A (16-22) 相混發現 (POG)7A 並無抑制的效果，而 A(POG)6、 A(POG)7 有抑制 A 聚集產生的效果，而這個效果並不完全來自於膠原蛋白的三股螺旋結構而可能與 PPⅡ 結構有關係。

Abstract
Alzheimer's disease ( AD ) is one of the most fatal neurodegenerative disease .The
major pathogenesis of Alzheimer's disease ( AD ) is neurofibrillary tangles and enile plaques , and the aggregation of the A 40 plays a crucial role in the pathogenesis of the senile plaques of Alzheimer's disease (AD) . Although the amyloid -protein has been studied extensively , the mechanisms of A aggregation in brain remain unclear . In this study , we investigate the influence of the collagen related peptides on A aggregation.
Here we chose the most important region of amyloid -protein responsible for aggregation , residues 16-22 (KLVFFAE) , as our study model . We incorporated the collagen sequence (POG)n into Aβ(16-22) and synthesized eight peptides:
WT-A(16-22) , A(16-22)-(POG)6 , A(16-22)-(POG)7, A(16-22)-(POG)10 , (POG)7-A(16-22) and (POG)10-A(16-22).Two collagen peptides (POG)7 and (POG)10 were synthesized as the control peptides for comparison.We used fluorescence spectroscopy, TEM , CD , FT-IR and DLS to study whether the collagen sequence can inhibit A aggregation .
CD measurements indicate that the Aβ(16-22) sequence at the N-terminus of (POG)n does not stabilize the triple-helical structure of collagen and such peptides have a weaker thermal stability than (POG)n .In contrast,when the Aβ(16-22) sequence is attached to the C-terminus of (POG)n, it can stablize the triple-helical structure of collagen.
From aggregation studies, we found that the amyloid aggregation did not occur when (POG)7 was incorporated into A(16-22) but the aggregation was enhanced when (POG)10 incorporated into A(16-22).The mixing experiments indicate that (POG)7A cannot inhibit the aggregation of WT-A(16-22) while A(POG)6 and A(POG)7 can.The inhibition effect may not be due to the triple-helical structure of collagen but the PPⅡ structure.

目錄
中文摘要 1
Abstract 2
謝誌 3
第 1 章 緒論 13
1-1 蛋白質結構簡介 13
1-2 貝它類澱粉蛋白(-amyloid，A ) 14
1-3 AD (Alzheimer's disease) 的介紹 15
1-4 貝它類澱粉蛋白 (beta-amyloid protein，A) 的介紹 16
1-5 貝它類澱粉蛋白 (A) 的結構介紹 18
1-6模擬 A(16-22) 胜肽的設計 21
1-7 膠原蛋白 (Collagen)簡介 26
1-8 膠原蛋白的結構 27
1-8-1 脯胺酸 (Pro) 穩定膠原蛋白的能力 27
1-8-2 氫氧脯胺酸 (Hyp) 穩定膠原蛋白的能力 29
1-9 微波固相胜肽合成法(microwave-assisted SPPS)介紹40, 41 33
1-9-1將第一個胺基酸接在樹脂上 (attachment of first residue to the resin) 36
1-9-2 去保護 (Deprotection) 36
1-9-4 耦合 (Coupling) 38
1-9-5 切除 (Cleavage) 38
1-10 螢光光譜 (Fluorescence spectroscopy)45 39
1-11 圓二色光譜 (Circular dichroism spectroscopy, CD)47 42
1-12 霍氏轉換紅外光譜儀（FT-IR）的介紹46 46
1-12-1 Michelson 干涉儀基本原理 47
1-13 動態光散射 (dynamic light scattering, DLS) 介紹 48
1-14-1 原理 51
1-14-2 儀器 52
1-15 研究動機 54
第 2 章 實驗部分 56
2-1 儀器 56
2-2 使用藥品 57
2-3 實驗步驟流程 61
2-4 合成 Fmoc-Pro-Hyp-Gly-OH tripeptide37 62
2-4-1 Boc-(4R,2S)-hydroxyproline (1) 之合成54 62
2-4-2 Boc-Hyp-Gly-OBn (2)之合成 63
2-4-3 Fmoc-Pro-Hyp-Gly-OBn(3)之合成 63
2-4-4 Fmoc-Pro-Hyp-Gly-OH(4)之合成 64
2-5 A 與膠原蛋白系列胜肽之合成 65
2-6 Incubation 條件 68
2-6-1 pH 7.0 緩衝溶液7: 68
2-6-2 50 mM 醋酸水溶液: 69
2-6-3 A 40 之incubation conditon: 69
2-7 CD 光譜量測與資料處理 69
2-7-1 Far-UV CD 光譜 (Wavelength scans) 69
2-7-2 變溫 CD 光譜量測 (Thermal denaturation) 70
2-7-3 變溫實驗資料處理24, 58 70
2-7-4 測量摺疊速率 ( Refolding kinetics) 72
2-7-5 動力學實驗資料處理 24 73
2-8 螢光光譜儀量測 7 74
2-9 量測動態光散射光譜 74
2-10 穿透式電子顯微鏡量測 75
2-11 量測霍氏轉換紅外光譜儀 75
第 3 章 實驗結果與討論 76
3-1 pH 7.0 下之A(POG)7 與 (POG)7A 系列胜肽探討 76
3-2 在 pH 7.0 下之A(POG)10 與 (POG)10A 系列胜肽探討 79
3-3 50 mM醋酸水溶液中膠原蛋白系列胜肽探討 80
3-4 胜肽摺疊速率的比較 84
3-5 在37 oC下 CD 光譜量測 86
3-6 A (16-22) 與膠原蛋白系列胜肽的螢光光譜量測與探討 87
3-7 A(POG)6結構之探討 89
3-8 利用穿透式電子顯微鏡觀察膠原蛋白與 A(16-22) 系列胜肽的結構形貌 91
3-9 WT-A (16-22) 和含有 (POG)7系列胜肽及 A(POG)6 作用探討 96
3-9-1 WT-A(16-22) 和含有 (POG)7系列胜肽及 A(POG)6 相混後 ThT 螢光結果探討 96
3-9-2 WT-A (16-22) 和含有 (POG)7系列胜肽及 A(POG)6 相混後CD結果探討 98
(A) (B) 98
3-9-3 WT-A (16-22) 和含有 (POG)7系列胜肽及 A(POG)6 TEM觀察結果 99
100
3-9-4 量測動態光散射光譜交叉比對 104
3-9-5 霍氏轉換紅外光譜(FT-IR) 105
3-10 A(POG)7 和 A(POG)6 與A 40 作用探討 108
第 4 章 結論 112
參考文獻 ( References ) 114

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