溫度敏感型胺基酸水膠包覆胰腺β細胞在組織工程上之應用

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研究生：	李俐瑩 Li, Li-Ying
論文名稱：	溫度敏感型胺基酸水膠包覆胰腺β細胞在組織工程上之應用 Thermosensitive Polypeptide Hydrogel encapsulated Pancreatic β cell for Tissue Engineering Application
指導教授：	朱一民 Chu, I-Ming
口試委員:	黃振煌 Huang, Chen-Huang 林世傑 Lin, Shih Chieh
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	57
中文關鍵詞：	胺基酸水膠、溫度敏感型、原位成膠、胰島素
外文關鍵詞：	polypeptide hydrogel, temperature sensitive, in situ gelation, insulin
相關次數：	點閱：3 下載：0
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　　本研究將甲基化聚乙二醇 (methoxy-poly(ethyleneglycol), mPEG) 之 -OH端改質成 -NH2，再共聚合上左旋丙胺酸 (ʟ-alanine)，形成一兩嵌段共聚物mPEG-polyalanine (mPEG-PA)，作為可應用於原位成膠之溫度敏感型胺基酸水膠。希望藉由材料之親疏水性質，提供良好之生長環境來促使細胞貼附，並藉由分析膠體性質及成膠機制，探討此膠體做為移植細胞的支架材料之潛力。
　　將此溫度敏感型胺基酸水膠以(_^1)H核磁共振光譜儀 ((_^1)H-NMR)、傅立葉轉換反射式紅外線光譜儀 (ATR-FTIR)、凝膠滲透層析儀 (GPC)、流變儀與掃描式電子顯微鏡 (SEM) 分析其各種性質及成膠溫度。接著探討材料對細胞毒性、水膠與細胞共培養之生物相容性，由MTT assay結果可以看到細胞存活率皆維持在90 % 以上；再來則進行胰腺 β cell功能性之體外測試，藉由酵素免疫分析法 (ELISA) 及即時定量聚合酶鏈式反應 (qRT-PCR) 確認β cell在包覆於水膠後，仍具有胰島素分泌的功能。
　　本研究最終期望開發一溫度敏感型胺基酸水膠，具有可注射、原位成膠以及良好之生物相容性等特性，成為具有優勢之組織工程的細胞支架，並使用此溫度敏感型胺基酸水膠mPEG-poly(alanine)作為胰島皮下移植的封裝材料，成為糖尿病治療之手段。

　　In this study, we report the synthesis and characterization of a temperature-sensitive polypeptide hydrogel. The -OH end of methoxy-poly(ethylene glycol) (mPEG) was modified to -NH2. Then, ʟ -alanine was co-polymerized with mPEG at the end of N-terminal of mPEG to form a two-block copolymer mPEG-poly(alanine) (mPEG-PA). The hydrogel can be used for in-situ gelation. After that, we explores the potential of this hydrogel as a cell scaffold application by analyzing gelation properties.
　　On the basis of the result of (_^1)H nuclear magnetic resonance spectrometer ((_^1)H-NMR) , Fourier transform reflective infrared spectrometer (ATR-FTIR), gel permeation chromatography (GPC), sol-to-gel transition, rheometer and scanning electron microscope (SEM) studies, the molecular weight and gelation properties of the hydrogel are determined. Furthermore, MIN6 β-cells were encapsulated in the hydrogel to observe the cell viability. By MTT assay, the cell viability may be reached above 90%. In order to confirm the insulin secretion, pancreatic β cells in vitro were demonstrated by enzyme immunoassay (ELISA) and quantitative real time polymerase chain reaction (qRT-PCR).
　　Overall, we show a strategy to modify mPEG hydrogel with bioactive polypeptide and use the thermosensitive methoxy-poly(ethylene glycol)- poly(alanine) (mPEG-PA) hydrogel as cell-encapsulating materials for the subcutaneous transplantation of pancreatic β cells. The thermosensitive hydrogel will be looked forwarded to act as a superior cell scaffold for local treatment.

摘要    i
Abstract    ii
致謝    iii
目錄    iv
圖目錄    vii
表目錄    ix
壹 文獻回顧與探討    1
1水膠介紹    1
1-1水膠的類型與特性    1
1-2水膠在組織工程上之應用    5
2胰臟與糖尿病    8
2-1胰臟的基本功能    8
2-2胰島素的分泌與作用    9
2-3糖尿病的病理機制    12
2-4糖尿病現行療法    14
貳 研究動機與目的    19
參 研究方法及步驟    20
1實驗藥品    20
2實驗器材    21
3 mPEG-poly(ʟ-alanine)嵌段共聚物製備    22
3-1 mPEG末端改質(-OH基→-NH2基)    22
3-2丙胺酸環化反應(N-carboxyl anhydride of ʟ-alanine, NCA-Ala)    23
3-3 mPEG-poly(ʟ-alanine)開環聚合反應    23
4 mPEG-poly(ʟ-alanine)嵌段共聚物之鑑定分析    24
4-1 核磁共振光譜儀(1H-NMR)    24
4-2 凝膠滲透層析儀(Gel Permeation Chromatography, GPC)    25
4-3 溶膠-凝膠相轉變測試(Sol-gel transition)    25
4-4 流變儀(Rheology)    25
4-5 掃描式電子顯微鏡(Scanning Electron Microscopy, SEM)    26
5 水膠之生物相容性(Biocompatibility of hydrogel)    26
5-1 細胞培養(Cell culture)    26
5-2 水膠與細胞共培養(Cell cultured with hydrogel)    27
6 細胞之功能性體外測試(Functional test of cells in vitro)    29
6-1 酵素免疫分析法(Enzyme-linked immunosorbent assay, ELISA)    29
6-2 定量即時聚合酶鏈式反應(Quantitative Real Time Polymerase Chain Reaction, qRT-PCR)    30
肆 實驗結果與討論    33
1 mPEG-poly(ʟ-alanine)嵌段共聚物之鑑定分析    33
1-1 共聚物單體結構分析    33
1-2 共聚物分子量分析    34
1-3 溶膠-凝膠相轉變測試    35
1-4 流變性質測試    36
1-5 共聚物三維結構分析    37
2水膠生物相容性之培養分析    38
3細胞之功能性體外測試    40
3-1 胰島素分泌能力    40
3-2 胰島素分泌相關基因表現    43
伍 結論    46
六 未來展望    48
陸 參考文獻    51
附錄    55


                                

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