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研究生:毛馨儀
研究生(外文):Hsin-Yi Mao
論文名稱:製造去細胞脂肪組織/海藻酸鈉微球包覆豬的脂肪幹細胞於組織工程的應用
論文名稱(外文):Fabrication of decellularized adipose tissue/alginate composite microspheres with pASCs encapsulation for tissue engineering
指導教授:游佳欣
指導教授(外文):Jiashing Yu
口試委員:蔡偉博陳賢燁劉澤英
口試委員(外文):Wei-Bor TsaiHsien-Yeh ChenTse-Ying Liu
口試日期:2015-07-22
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:99
中文關鍵詞:脂肪幹細胞去細胞的脂肪組織海藻酸鈉微載體周邊動脈疾病
外文關鍵詞:Adipose derived stem cells (ASCs)Decellularize adipose tissue(DAT)AlginteMicrospheresPAD
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周邊動脈疾病是一種不可忽視的疾病,近期治療方法使用對於組織的再生和血運重建,植入幹細胞療法是一個具有極大潛力的治療方式。幹細胞療法的目標是更換並修復或加強受損組織或器官的功能。脂肪幹細胞ASCs因具分化能力,有相較於其他幹細胞更為方便的萃取方法和充足的來源等優點,為一在組織工程和在生醫學領域上新興的幹細胞來源;然而,有兩個問題是在細胞移植或異種移植前需要解決的主要問題,如此一來才可以達到臨床應用所能接受的標準:控制免疫排斥反應以及控制植入的細胞其增殖的情形。
在本研究中,我們著重於去細胞的脂肪組織(DAT),這是一種天然細胞外基質(ECM)。我們相信,從生物體本身提取的生物材料有很大的潛力作為支架材料的幹細胞培養,以及幫助組織重建,特別是天然的細胞外基質和幹細胞都是來自脂肪組織。我們將利用去細胞組織的溶液(DATsol)與海藻酸鈉的材料對於脂肪幹細胞ASCs的影響。
在這項實驗中,我們萃取DATsol並混合有高、低兩種分子量的海藻酸鈉培養脂肪幹細胞ASCs,進行了許多實驗研究。在二微的體外實驗研究表明,混有DATsol的海藻酸鈉可以成功促進脂肪幹細胞ASCs的貼附和分化;在混有DATsol的海藻酸鈉固定化脂肪幹細胞ASCs方面,顯示出混有DATsol之海藻酸鈉包覆細胞會使得細胞有較高的存活率,整體存活率都高於80%。我們認為使用脂肪幹細胞作為治療下肢缺血患者因病情嚴重而受到影響的病症發展,是個有潛力成為一種成熟且穩定的幹細胞治療方式。


Cell-based therapies, new and current regenerative treatment, can use stem cells to create living and functional tissues to regenerate and repair tissue and organs in the body. In peripheral arterial disease, it has been demonstrated that stem cell treatment can improve perfusion and stimulate neovascularization. However, it usually suffers from some deficiencies, like immune rejection and limited proliferation of implanted cells.
In this study, we focus on the application of the decellularize adipose tissue (DAT), which is a native extracellular matrix (ECM). We believe that the biomaterial extracted from the organism itself have great potential as a scaffold materials for stem cell culture as well as new tissue construct formation, especially the native ECM and stem cell are from the same origin (adipose tissue). We used DATsol (DAT solution) combined with alginate to culture Adipose stem cells (ASCs).
In this work, we extracted DATsol to mix in high and low molecular weight alginate and investigated. The 2-D in virto study of DATsol/alginate could promote cell adhesion and differentiation; ASCs, immobilized in DATsol/alginate microspheres, showed metabolic activity which with an overall viability higher than 80%.
We believe that the use of adult stem cell therapy in ischemia patients allows for the development of new mature and stable capillaries in patients affected by the severe condition.


誌謝.................................................................................................................................. 1
摘要.................................................................................................................................. 2
Abstract ............................................................................................................................. 3
圖目錄.............................................................................................................................. 7
表目錄.............................................................................................................................. 9
第一章 介紹.................................................................................................................. 10
1-1 組織工程......................................................................................................... 10
1-1-1 細胞.................................................................................................... 10
1-1-2 支架.................................................................................................... 12
1-1-3 訊息因子............................................................................................ 14
1-2 細胞固定化..................................................................................................... 14
1-3 海藻酸鈉......................................................................................................... 15
1-3-1 海藻酸鈉的凝膠性質........................................................................ 15
1-3-1 海藻酸鈉的表面性質........................................................................ 16
1-4 天然材料-去細胞脂肪組織........................................................................... 17
1-4-1 去細胞組織的成分及特性................................................................ 17
1-4-2 去細胞................................................................................................ 19
1-5 利用空氣動力機包埋細胞............................................................................ 22
1-6 周邊動脈疾病(peripheral artery disease)...................................................... 23
1-6-1 周邊動脈疾病治療方法.................................................................... 24
1-6-2 幹細胞治療周邊動脈疾病的潛能................................................... 25
第三章材料與方法 ...................................................................................................... 27
3-1 實驗設備........................................................................................................ 27
3-2 實驗藥品........................................................................................................ 29
5
3-3 實驗步驟........................................................................................................ 32
3-3-1 去細胞脂肪組織蛋白質溶液的萃取................................................ 32
3-3-2 去細胞的程度分析........................................................................... 34
3-3-3 豬的脂肪幹細胞培養與保存........................................................... 37
3-3-4 2-D 細胞培養於DATsol/海藻酸鈉的複合材料上..................... 40
3-3-5 脂肪幹細胞在DATsol/海藻酸鈉膜上的分化情形........................ 46
3-3-6 微囊固定化脂肪幹細胞.................................................................... 53
3-3-7 動物實驗........................................................................................... 56
第四章結果與討論 ...................................................................................................... 60
4-1 去細胞脂肪組織............................................................................................. 60
4-1-1 去細胞脂肪組織(DAT)..................................................................... 60
4-1-2 去細胞脂肪組織蛋白質溶液萃取(DATsol) .................................... 62
4-1-3 去細胞前後的GAGs 含量................................................................ 63
4-1-4 去細胞前後膠原蛋白含量................................................................ 64
4-2 豬的脂肪幹細胞培養..................................................................................... 65
4-3 二維細胞培養於DATsol/海藻酸鈉的複合材料........................................ 66
4-3-1 DATsol/海藻酸鈉的複合材料貼附測試.......................................... 66
4-3-2 DATsol/海藻酸鈉的複合材料增生測試.......................................... 69
4-3-3 ASCs 之脂肪細胞分化能力測定..................................................... 73
4-3-4 ASCs 之骨細胞分化能力測定......................................................... 75
4-3-5 ASCs 的體外基因表現量分析......................................................... 78
4-4 微囊固定化脂肪幹細胞................................................................................ 81
4-4-1 海藻酸鈉微囊球粒徑測試............................................................... 81
4-4-2 海藻酸鈉微囊球計細胞活性........................................................... 82
4-5 動物實驗........................................................................................................ 86
第五章 結論 .................................................................................................................. 90
參考文獻........................................................................................................................ 92
口試問答........................................................................................................................ 97


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