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研究生:鄭琬蓉
研究生(外文):Cheng, Wan-Jung
論文名稱:利用組胺酸-金屬配位作用來進行膠原蛋白自組裝之探討
論文名稱(外文):Self-Assembly of Collagen-Related Peptides by Metal-Histidine Coordination
指導教授:洪嘉呈洪嘉呈引用關係
指導教授(外文):Horng, Jia-Cherng
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系
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:2013
畢業學年度:101
語文別:英文
論文頁數:86
中文關鍵詞:膠原蛋白組胺酸金屬配位纖維自組裝
外文關鍵詞:collagenhistidinecoordinationfiberself-assembly
相關次數:
  • 被引用被引用:1
  • 點閱點閱:132
  • 評分評分:
  • 下載下載:15
  • 收藏至我的研究室書目清單書目收藏:0
膠原蛋白大量存在於哺乳類體內,因為其本身即存在於生物體內,膠原蛋白具有生物降解性、生物相容性、生物吸收性、與生物活性物質協同作用、止血、可轉化成不同形式、生物降解的可調性等優異的特性,所以已被廣泛應用於生醫材料上。目前膠原蛋白產品的製作,是從動物組織中萃取出膠原蛋白纖維,與人類的膠原蛋白結構略有差異,在應用上須注意免疫方面的問題,所以尋找有效的製備方法使合成的短模擬膠原蛋白胜肽能夠進行自組裝形成類似天然膠原蛋白的高階結構是許多團隊的研究方向。
在本實驗中組胺酸被引入模擬膠原蛋白胜肽,我們合成三條短膠原蛋白胜肽:HG(POG)9GH、HG(POG)4PHG(POG)4GH和GG(POG)9GG。將胜肽與含有金屬離子的溶液混合,藉由組胺酸與金屬離子的配位關係誘發膠原蛋白胜肽自組裝形成高階超分子結構。所合成出來的胜肽先利用遠紫外光圓二色光譜來確定其結構,進行變溫實驗來測試其穩定度,利用動態光散射量測來觀察胜肽在水溶液中的大小,也使用掃描式電子顯微鏡和穿透式電子顯微鏡來觀察胜肽自組裝生成的超分子結構形狀。
我們發現所得到的超分子結構可藉由在不同時間點加入金屬來控制其生長形狀,此外也發現此三條胜肽在沒有金屬的環境下,仍可自組裝形成微米尺度的結構,而且含有組胺酸的胜肽和沒有組胺酸的對照組所生長的形狀不同,我們推測影響其生長形狀的不同為置入序列中的組胺酸所影響,所以進行了改變溶液酸鹼性的實驗,來探討組胺酸對膠原蛋白模擬胜肽自組裝的影響。
雖然膠原蛋白自組裝的過程仍不清楚,但是在我們的研究當中發現膠原蛋白自組裝的速度、外加因素的影響時機,還有組胺酸的引入,皆對膠原蛋白自組裝的結果有明顯的影響,希望這樣的結果能對未來的膠原蛋白自組裝的設計能有更多的了解與幫助。

Collagen is a biodegradable and biocompatible material, and has been applied in medical uses for decades. However, animal-derived collagens have several drawbacks, such as low thermal stability, nonspecific cell adhesion, and antigenicity. To solve these problems, preparing collagen-related biomaterial from short mimetic collagen peptides has received many attentions and become an emerging research topic. Our previous studies have shown that His-metal coordination can induce unstable short mimetic collagen peptides to assemble into a higher order structure.
In this work, we prepared three collagen related peptides (CRPs): HG(POG)9GH, HG(POG)4PHG(POG)4GH, and GG(POG)9GG, of which two peptides contain His residues, to study their assembled structures. The size and topology of results show that His-metal coordination can promote mimetic collagen peptides to form macro-scale structures, and the topologies depend on metals and the time of adding metal ions into peptide solutions. Circular dichroism spectroscopy was used to examine the structure and the thermal stability of collagen mimetic peptides. Dynamic light scattering (DLS), SEM, and TEM were used to assess the size and the topology of the assembled structures. The CRPs in this work can form microstructures without the assistance of metal ions. Thus, pH dependent assembly of these CRPs was also examined.
Although we are not able to clarify the process of self-assembly at the present stage, we did find the impact of the rate of self-assembly, His-metal coordination, and the His-His interaction on the assembly of CRPs. Our results may be useful and helpful for the future development of collagen-related materials.

中文摘要 I
Abstract II
Table of contents III
List of Figures VI
List of Schemes VIII
List of Tables IX
Chapter1 Introduction 1
1.1 Collagen 1
1.2 Structure of the collagen triple helix 4
1.2.1 History of collagen structure 4
1.2.2 Pro-Hyp-Gly trimer 6
1.2.3 Substitution in collagen related peptides 7
1.2.4 Natural collagen structure 8
1.2.5 Self-assembly of collagen 9
1.3 Metals in biological system 13
1.3.1 Essemtial chemical elements33,34 13
1.3.2 Hard-soft acid-base theory (HSAB)34 16
1.3.3 Biological ligands for metal ions34 17
1.3.4 Metal-triggered collagen self-assembly 19
1.4 Motivation and Purpose 21
Chapter2 Methods and materials 23
2.1 Equipment 23
2.2 Reagents 24
2.3 Circular dichroism Spectroscopy, CD 26
2.4 Dynamic light scattering, DLS39,40 30
2.5 Solid phase peptide synthesis, SPPS 32
2.5.1 Attachment of the first amino acid to resin 34
2.5.2 Deprotection 34
2.5.3 Activation 35
2.5.4 Coupling 36
2.5.5 Cleavage 36
2.6 Fmoc-Pro-Hyp-Gly-OH synthesis 37
2.6.1 Boc-Hyp-OH synthesis 37
2.6.2 Boc-Hyp-Gly-OBn synthesis 38
2.6.3 Fmoc-Pro-Hyp-Gly-OBn synthesis 38
2.6.4 Fmoc-Pro-Hyp-Gly-OH synthesis 39
2.7 SPPS of collagen related peptides 41
2.8 Metal-His coordination 42
2.8.1 Collagen mimetic peptide Y9, HY9, and metal solution preparation 42
2.8.2 Circular dichroism 42
2.8.3 Dynamic light scattering 43
2.8.4 Scanning electron microscope, SEM 43
2.8.5 Transmission electron microscope, TEM 44
2.9 CD data analysis 44
2.9.1 Thermal denaturation 44
2.9.2 Kinetic experiments 47
Chapter3 Results and Discussion 48
3.1 Metal-triggered CRPs self-assembly 48
3.1.1 CD spectroscopy 48
3.1.2 Thermal denaturation 50
3.1.3 DLS 53
3.1.4 SEM 56
3.1.5 TEM 64
3.2 pH-dependent experiments 67
3.2.1 Far-UV CD 67
3.2.1 Thermal denaturation 69
3.2.2 Kinetic experiment 71
3.2.3 DLS 74
3.2.4 SEM 76
Chapter4 Conclusion 80
Reference 82
Appendix 86

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