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研究生:李政昕
研究生(外文):Cheng-Hsi Li
論文名稱:脂肪組織中獲取具全能性幹細胞:應用在聚乳酸-聚乙醇酸共聚物三度立體支架之骨組織工程
論文名稱(外文):Multipotent Stem Cells from Adipose Tissue :Application forPLGA 3D Scaffold in Skeletal Tissue Engineering
指導教授:蕭世裕
指導教授(外文):Shyh-Yu Shaw
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:106
中文關鍵詞:脂肪幹細胞骨組織工程
外文關鍵詞:adipose-derived stem cellskeletal tissue engineering
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組織工程是研究生物組織與材料相互關係的一門學問,運用細胞移植的技術作細胞治療(Cell Therapy)更是相當熱門的研究,其中,幹細胞特殊具有的高度分化能力,希望能夠在體外大量培養幹細胞,運用特殊的因子與合適的環境,即可誘導其分化成所需要的組織。
本研究論文中所使用的幹細胞,是從脂肪組織中取得具有分化能力的脂肪幹細胞,具有取得容易、細胞數目多、分化能力佳且不會引起道德爭議等優點。經本實驗證明人類脂肪幹細胞分化成骨細胞、軟骨細胞、肌肉細胞和脂肪細胞的比例分別為:81.62%、41.33%、1.75%與25.49%。此外,本研究利用了生物可降解性的生醫材料聚乳酸-聚乙醇酸共聚物PLGA (poly DL-lactic-co-glycolic acid)作為三度立體支架,研究PLGA對於脂肪幹細胞的生物相容性、黏著程度、生長速率以及分化的影響,研究結果顯示脂肪幹細胞在孔徑300∼500um的多孔性PLGA上,可以得到較好的生長與分化結果。
Tissue engineering is a new field in biotechnology and has a powerful potential for tissue regeneration. Cell is a major component of tissue engineering, and stem cell with good differentiation capacity will be the most ideal source of cells. Human adipose tissue, obtained by suction-assisted lipectomy (i.e. liposuction), was processed to obtain a fibroblast-like population of cells named processed lipoaspirate (PLA cells) or adipose-derived stem cells(ADSCs). In this research, we were able to induce the differentiation of ADSCs into mesenchymal cell lineages, such as osteoblast, chrondrocyte, myocytes and adipocytes with the differentiation ratio of 81.62%, 41.33%, 1.75%, and 25.49%, respectively. In addition, we examined the attachment, growth and differentiation ability of ADSCs in porous PLGA (poly DL-lactic-co-glycolic acid). The result indicates that the proliferation & differentiation abilities of ADSCs cells is better in large pore size PLGA (300 ~ 500 um).
中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii
誌謝……………………………………………………………………iii
表目錄…………………………………………………………………viii
圖目錄…………………………………………………………………ix
第一章 緒論……………………………………………………………1
一、組織工程簡介………………………………………………………4
1- 1組織工程的進程……………………………………………………4
1- 2骨組織修復…………………………………………………………6
1- 2- 1骨骼系統之重要性與常見傷害…………………………………6
1- 2- 2骨組織工程的發展………………………………………………8
二、先驅細胞的取得、培養與誘導分化………………………………11
2- 1 先驅細胞的定義…………………………………………………11
2- 1- 1幹細胞的來源與分類…………………………………………12
2- 1- 2 幹細胞的應用…………………………………………………14
2- 1- 3 脂肪幹細胞的特性(The character of ADSCs) ………………15
2- 2 生長與分化因子(Growth factor & differentiation factor) ………18
2- 2- 1 與脂肪細胞分化相關之生長因子……………………………20
2- 2- 2 與骨細胞分化相關之生長因子………………………………23
2- 2- 3 與軟骨細胞分化相關之生長因子……………………………27
2- 2- 4 與肌肉細胞分化相關之生長因子……………………………28
三、生醫材料……………………………………………………………30
3- 1 生醫材料的應用…………………………………………………30
3- 1- 1 生醫材料所需的基本條件……………………………………31
3- 1- 2 生醫材料的種類………………………………………………32
3- 1- 3 高分子生醫材料………………………………………………34
四、研究目的……………………………………………………………35
第二章 實驗…………………………………………………………36
一、實驗器材與實驗用藥品……………………………………………36
二、實驗步驟……………………………………………………………39
2- 1 脂肪幹細胞的萃取與培養………………………………………39
2- 1- 1 細胞萃取步驟Stromal Vascular Fraction(SVF)……………39
2- 2 脂肪幹細胞的誘導分化(Differentiation of ADSCs)…………41
2- 2- 1 脂肪細胞的誘導分化(Adipogenesis)………………………42
2- 2- 2 骨細胞的誘導分化(Osteogenesis)…………………………44
2- 2- 2- 1 骨細胞的誘導分化比率(Osteogenesis ratio)……………48
2- 2- 2- 2 繼代後對於骨細胞的誘導分化能力的影響(Passage effect on Osteogenesis ability)……………………………………………………49
2- 2- 3 軟骨細胞的誘導分化(Chondrogenesis)……………………49
2- 2- 4 肌肉細胞的誘導分化(Myogenesis)…………………………51
2- 5 PLGA試片的製備…………………………………………………56
2- 6 細胞在PLGA上誘導成骨細胞…………………………………56
2- 6- 1 細胞植入PLGA支架…………………………………………57
2- 6- 2 細胞植入PLGA支架的效率(Seeding efficiency)…………57
2- 6- 3 細胞於PLGA支架上的生長(Proliferation of ADSCs on PLGA)…………………………………………………………………59
2- 6- 4 組織切片觀察細胞生長情形…………………………………60
2- 6- 5 細胞於PLGA支架上的分化(Differentiation of ADSCs on PLGA)…………………………………………………………………61
第三章 結果與討論…………………………………………………64
一、脂肪幹細胞ADSCs的取得與分化特性之探討…………………64
1- 1 脂肪幹細胞ADSCs之取得與其生長曲線………………………64
1- 2 脂肪幹細胞ADSCs之分化能力測定……………………………65
1- 2- 1 脂肪幹細胞ADSCs之骨細胞分化能力測定…………………66
1- 2- 2 繼代與長時間培養對於ADSCs之骨細胞分化能力影響之測定………………………………………………………………………67
二、脂肪幹細胞ADSCs在不同孔徑之PLGA上生長與分化特性之探討………………………………………………………………………68
2- 1 脂肪幹細胞ADSCs之植入效率測定……………………………68
2- 2 脂肪幹細胞ADSCs於PLGA上生長測定………………………69
2- 3 脂肪幹細胞ADSCs於PLGA上分化成為骨細胞的測定………70
第四章 結論…………………………………………………………73
第五章 參考文獻……………………………………………………76
附錄……………………………………………………………………105
表、實驗中所用到的組織染色………………………………………105
自述……………………………………………………………………106
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