臺灣博碩士論文加值系統

由於胜肽的高選擇性，可由不同氨基酸組成的獨特性和可變性以及與蛋白質相比更易於生產的優點，最近胜肽的開發和應用受到了科學家的極大關注。在這裡，本研究開發有別以往胜肽與聚合物合成的方法和提供了兩種胜肽-聚合物偶聯物的生物活性材料。在第一部分中，固相胜肽合成(Solid phase peptide synthesis)用於合成由賴氨酸丙烯醯胺(Lysine acrylamide)和常規氨基酸組成的丙烯酸胜肽(acrylic peptides)，來作為水凝膠的交聯劑，當使用胰蛋白酶處理時，胜肽可以被切割，開發出具有酶促降解的水凝膠。與N，N'-亞甲基雙丙烯醯胺(N,N'-Methylenebisacrylamide)交聯劑形成的水凝膠相比，發現在經過胰蛋白酶浸泡後，只有丙烯酸胜肽水凝膠會崩解，這在傷口敷料和藥物釋放應用上有很大的潛力。工作的第二部分透過6-Acrylamidohexanoic Acid來製備丙烯酸胜肽並與精氨酸-甘氨酸-天冬氨酸（RGD）的整合素(Integrin)識別配體結合在一起。RGD胜肽為一種細胞黏附序列，因為它易於與整合素相互作用。在這裡，將帶有丙烯酸胜肽-RGD配體的水凝膠和一般水凝膠在DMEM中與細胞共培養。以直立顯微鏡觀察水凝膠表面，結果表明具有丙烯酸胜肽-RGD配體的水凝膠有細胞黏附，與一般的水凝膠不同，透過丙烯酸胜肽-RGD配體的加入，可使原本不利於細胞生長的材料能支持細胞生長而更貼近體內條件，此可應用於修復功能性生物材料上。最後，利用衰減全反射傅立葉轉換紅外線光譜(ATR-FTIR)結構鑒定和X射線光電子能譜儀(XPS)表面元素分析來確認丙烯酸胜肽-RGD配體與水凝膠的聚合，和透過丙烯酸胜肽-RGD配體與螢光分子聚合成線性螢光高分子來標記細胞，證實丙烯酸胜肽-RGD配體與聚合物形成線性高分子時仍能與細胞作用。本研究開發了胜肽-聚合物偶聯物和其合成的策略，期望能將此應用於醫療方面並提供新的生物材料設計方法。

Due to the high selectivity of peptides, the uniqueness and variability that can be composed of different amino acids, and the advantages of easier production compared with proteins, the development and application of peptides have recently received great attention from scientists. Here, this study provides a method different from the previous synthetic methods of peptides and polymers and provides two peptide-polymer conjugates for bioactive polymeric materials. In the first part, solid phase peptide synthesis was applied for synthesizing acrylic peptides composed of Lysine acrylamide and conventional amino acids, as a crosslinker for hydrogels, then use nuclear magnetic resonance (NMR) and mass spectrometry (MS) to identify its chemical structure. The peptide can be cleaved when treating with trypsin, and the hydrogel with enzymatic degradation has been developed. Compared with the hydrogel formed by N,N'-Methylenebisacrylamide crosslinker, we found that after soaking in trypsin, only the acrylic peptide hydrogel disintegrated. The applications in biomimetic polymers such as biodegradable scaffolds and drug delivery has great potential. The second part of the work is to prepare acrylic peptides through 6-Acrylamidohexanoic Acid and combine them with the Integrin recognition ligand of arginine-glycine-aspartic acid (RGD). RGD peptide is a cell adhesion sequence because it is easy to interact with integrins. Here, the hydrogel with acrylic peptide-RGD ligand and general hydrogel are co-cultured with cells in DMEM. Observing the surface of the hydrogel with the optical microscope, the results presented that the hydrogel with acrylic peptide-RGD ligands has cell adhesion. Different from general hydrogels, the addition of acrylic peptide-RGD ligand can support cell growth with materials that are not conducive to cell growth, and closer to the conditions in vivo, this can be applied to the functional biomaterials for tissue repair. Finally, FT-IR structure identification and XPS surface element analysis were used to confirm the polymerization of acrylic peptide-RGD ligand and hydrogel, and through the polymerization of acrylic peptide-RGD ligand and fluorescent molecules into linear fluorescent polymers to label cells, confirming that acrylic peptide- RGD ligand and polymer can still interact with cells when they form a linear polymer. This research has developed peptide-polymer conjugates and their synthesis strategies. It is hoped that this can be applied to medical treatment and provide new biomaterial design methods.

目錄
中文摘要....……....……………………………………………….……….……i
Abstract....……....………………………………………………………….….ii
目錄....……....………………………………………………………………….iv
圖目錄....……....…………………………………………………………...….ix
表目錄....……....………………………………………………………….…...xi
第一章 文獻回顧………………….......……....……………….…….………1
1-1 氨基酸………….………….…..…..……..……………….……………1
1-2 胜肽(Peptide)……..……...………….……………………………….…2
1-2-1胜肽的功能與發展………………………………….……...……….3
1-2-2 胜肽的應用.…………….…………...…………………………….5
1-2-3 固相胜肽合成(Solid-phase peptide synthesis，SPPS)……...…….6
1-2-4 胺基酸保護基(Amino Acid-Protecting Groups)………...…...……7
1-2-5 精甘天冬氨酸胜肽(RGD peptide)…………………………………8
1-2-6 整聯蛋白(Integrin)………...……………………………………….9
1-3 水凝膠(Hydrogel)….…..………………….……………………………9
1-3-1 水凝膠的聚合…………….….………..………………….…….…10
1-4 兩性離子(Zwitterion)…………………………………….....……...…11
1-4-1 2-甲基丙烯醯氧基乙基磷酸膽鹼(MPC)聚合物……..…...…....12
1-5 胜肽與聚合物的合成…….…….…………………………….………13
1-5-1 胜肽-聚合物聚合方法之比較…..……………………………….18
1-6 胜肽-聚合物偶聯物…………………………………………………..19
1-6-1 胜肽-聚合物偶聯物之研究..…………………………………….20
1-7 生物性材料……………………………………………………...……21
1-7-1 生物性材料的條件和應用……………………………………….21
1-8 基於胜肽的生物性材料……………………………………………...22
第二章 研究目的…………………………………………………..….……24
第三章 實驗藥品與設備及實驗方法…………..……..……….……….25
3-1 實驗藥品與設備……………………………….…..…………………25
3-2 材料合成………………………………………...……………………28
3-2-1 丙烯酸賴氨酸(Lysine acrylate acid)………….…….…………...28
3-2-2 9-芴甲氧羰醯氯-丙烯酸賴氨酸(FMOC-Lysine acrylate acid).29
3-2-3 2-甲基丙烯醯氧基乙基磷酸膽鹼水凝膠(2-Methacryloyloxyethyl. phosphorylcholine (MPC) Hydrogel)…..29
3-2-4 丙烯酸P5胜肽作為交聯劑之MPC水凝膠………………….30
3-2-5 丙烯酸P3-RGD胜肽之MPC水凝膠…………………………30
3-2-6 矽烷化玻璃製備…………………………………..……………31
3-2-7 丙烯酸P3-RGD胜肽之螢光高分子（Poly(MPC-r-P3-r-MTR）……………………………………………………………32
3-3 實驗方法……………..……………………….………………………33
3-3-1 選擇9-芴甲氧羰醯氯-丙烯酸賴氨酸(FMOC-LysAA)之沖堤劑………………………………………………..……..………...33
3-3-2 9-芴甲氧羰醯氯-丙烯酸賴氨酸(FMOC-LysAA)之純化……..34
3-3-3 FMOC-LysAA於高溫下在各時間的測試……………...……..35
3-3-4 DMEM配置……….……..…………………………….……….35
3-3-5 NIH-3T3細胞培養…….……………………….……………….36
3-3-6 液相層析質譜儀（LC-MS）………….....……..……………...36
3-3-7 衰減全反射傅立葉轉換紅外線光譜（ATR-FTIR）……….…37
3-3-8 X射線光電子能譜儀（X-ray photoelectron spectroscopy， XPS）…………………………………….………..……………37
3-3-9 丙烯酸P5胜肽水凝膠之降解測試………..….………..….….37
3-3-10 丙烯酸P3-RGD胜肽水凝膠之細胞貼附測試…………….….38
3-3-11 丙烯酸P3-RGD胜肽之螢光高分子標記細胞………………..38
3-3-12 統計分析….……………………………………………..…..…38
第四章 結果與討論…………………...………………...………………....39
4-1 Lysine acrylate acid之NMR光譜圖……...…………………………39
4-1-1 Lysine acrylate acid之質譜圖………………..……….…………40
4-1-2 Lysine acrylate acid之ATR-FTIR光譜圖………...……...…….40
4-2 FMOC-LysAA之NMR光譜圖……………………………………...41
4-2-1 FMOC-LysAA之質譜圖……………………………………..….42
4-2-2 FMOC-LysAA之ATR-FTIR光譜圖…………...………………43
4-3 FMOC-LysAA之總離子流（TIC）色譜圖….…………...………....43
4-3-1 FMOC-LysAA之提取離子（EIC）色譜圖…………......……...44
4-4 FMOC-LysAA薄層層析之沖堤劑…............................................…..45
4-4-1 FMOC-LysAA管柱層析之UV-Vis光譜圖…….……………...46
4-4-2 FMOC-LysAA純化前後之NMR光譜圖…………...….……....47
4-4-3 FMOC-LysAA純化前後之HPLC-質譜圖…………….....…..…48
4-5 FMOC-LysAA對於高溫環境下的影響…..….……….......................49
4-6 MPC水凝膠與丙烯酸P3-RGD胜肽水凝膠之FT-IR光譜圖…......50
4-7 MPC水凝膠與丙烯酸P3-RGD胜肽水凝膠之表面元素分析……..51
4-8 MPC水凝膠與丙烯酸P5胜肽水凝膠之降解測試比較……………53
4-9 MPC水凝膠與丙烯酸P3-RGD胜肽水凝膠之細胞貼附比較……..55
4-9-1 MPC水凝膠與丙烯酸P3-RGD胜肽水凝膠之細胞生長比較...57
4-10 Poly(MPC-r-P3-r-MTR)之NMR光譜圖…….…………...………...60
4-10-1 Poly(MPC-r-P3-r-MTR)與Poly(MPC-r-MTR)之NMR光譜圖.61
4-10-2 Poly(MPC-r-P3-r-MTR)與Poly(MPC-r-MTR)之FT-IR光譜 圖…...…………...….…………...….…………...….……….………...…62
4-11 丙烯酸P3胜肽之螢光高分子標記細胞..............................................63
第五章 結論…………………………………………………………………65
第六章 未來展望……………………………………...……………………66
第七章 參考文獻………….…………………………………..……………67

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