博碩士論文 102324032 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:11 、訪客IP:34.204.191.31
姓名 林哿塵(Ke-chen Lin)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究
(Pluripotency and Proliferation of Amniotic Fluid-Derived Stem Cells Cultured on Biomaterials Grafted with Oligopeptides and Cyclic RGD Having Optimal Elasticity)
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摘要(中) 人類羊水來源的幹細胞是一種多能性胎細胞,其具有分化成三胚層以及各種專門細胞系的能力,在再生醫學與組織工程中,從羊水中取得的幹細胞將是一種更適合的幹細胞來源,然而幹細胞的一些特質,例如適當的分化和多能性的維持,不僅會受到細胞自己本身的影響,也會因為周遭微環境的不同而改變,另一方面而言,細胞的多能性與分化將會受到用來培養的生醫材料之生物線索(例如細胞外間質)以及物理線索(例如表面硬度)的不同而影響其命運,我們在這篇研究中,利用將羊水幹細胞培養在具有不同軟硬度的聚- (乙烯醇共衣康酸) 膜 (PVA-IA films) 接枝某些細胞外間質的寡肽,並將其與環狀肽 (Cyclic RGD)作結合來探討羊水幹細胞的多能性維持與分化能力。我們從測量多能性基因 (Oct4、Sox2、Naong) 的結果中發現,羊水幹細胞培養在較軟的PVA-IA films (19.6 百帕) 接枝細胞外間質寡肽會有較高的多能性表現,而羊水幹細胞培養在較硬的PVA-IA films膜 (30.4 百帕)接枝第一型膠原蛋白寡肽和環狀肽,其會比在相同硬度之下只接枝第一型膠原蛋白寡肽還要有更高的多能性表現。我們從羊水幹細胞培養在以PVA-IA films為基質的材料且培養於細胞成長培養液中意外的發現,擁有較高多能性表現的細胞也會同時表現較高的前期三胚層分化基因,從這項結果可說明羊水幹細胞是一種非勻相的細胞系,其細胞組態包含了具有高多能性的幹細胞以及容易分化的幹細胞。在所有的結果中可發現,用來培養的生醫材料之物理效應 (例如表面硬度) 與生物效應 (例如細胞外間質) 的確可用來調控幹細胞的多能性和分化能力。
摘要(英) Human amniotic fluid-derived stem cells (AFSCs) are pluripotent fetal cells capable of differentiating into multiple lineages, including representatives of the three embryonic germ layers. Stem cells which derived from human amniotic fluid may become a more suitable source of stem cells in regenerative medicine and tissue engineering. However, stem cell characteristics, such as proper differentiation and maintenance of pluripotency, are regulated not only by the stem cells themselves but also by their microenvironment. On the other hand, the stem cell fates of pluripotency and differentiation can influence by biological cues (i.e. extracellular matrix (ECM)) and physical cues (i.e. elasticity) of cell culture biomaterials. Here we investigated the maintenance of pluripotency and differentiation ability of AFSCs cultured on poly(vinylalcohol-co-itaconic acid), PVA-IA films grafted with several ECM-derived oligopeptides and combined with cyclic RGD having different elasticity. AFSCs cultured on soft PVA-IA films (19.6 kPa) grafted with ECM-derived oligopeptides showed high pluripotency, as assessed by the pluripotent gene expression (Oct4, Sox2, and Naong). AFSCs grow on stiff PVA-IA films (30.4 kPa) grafted with the combination of oligopeptides derived from collagen type I (oligoCOL) and cyclic RGD peptide showed higher pluripotency than that grafted with oligoCOL. Surprisingly, AFSCs cultured on PVA-IA films showed higher pluripotency also expressed higher level of early differentiation markers for three germ layers (Nestin, Runx2, Sox17) in expansion medium. This result suggests that AFSCs are heterogenous and that this population contains highly pluripotent stem cells and stem cells that can be easily differentiated. It is suggested that physical cues such as stiffness of culture biomaterials and biological cues such as ECM-derived peptides can guide pluripotency and differentiation ability of stem cells.
關鍵字(中) ★ 羊水幹細胞
★ 聚乙烯醇
★ 硬度
★ 環狀肽
★ 寡肽
★ 生醫材料
關鍵字(英) ★ Amniotic fluid-derived stem cells
★ Polyvinylalcohol
★ Elasticity
★ Cyclic RGD
★ Oligopeptides
★ Biomaterials
論文目次 INDEX OF CONTENT
CHAPTER ONE INTRODUCTION 1
1-1 Stem cell 1
1-1-1 Potency of stem cells 2
1-1-2 Totipotency 2
1-1-3 Pluripotency 2
1-1-4 Multipotency 2
1-2 Souces of stem cells 3
1-2-1 Embryonic stem cells (ESCs) 3
1-2-2 Induced pluripotent stem cells (iPSCs) 5
1-2-3 Mesenchymal stem cells (MSCs) 6
1-3 Amniotic fluid-derived stem Cells 7
1-3-1 Amniotic fluid cells 7
1-3-2 Amniotic fluid cell type 9
1-3-3 Isolation of amniotic fluid stem cell (AFSCs) 11
1-3-4 Characterization of amniotic fluid stem cell 12
1-3-5 Pluripotency of amniotic fluid stem cells 14
1-3-6 Differentiation of amniotic fluid stem cells 16
1-4 Niches of stem cells 18
1-4-1 Soluble factors 19
1-4-2 Cell-cell interactions 19
1-4-3 Physical cues affect ex vivo expansion 20
1-4-4 Cell-biomaterials interactions 23
1-5 Extracellular-matrix (ECM) and ECM-mimicking oligopeptides 24
1-5-1 Type and classification of artificial ECMs 25
1-6 The effect of extracellular matrix (ECM) to stem cells 31
1-7 Markers of pluripotent gene 32
1-8 Markers of differentiation lineages of MSCs 34
1-9 Polymerase chain reaction (PCR) 36
1-9-1 Procedure of PCR 37
1-9-2 Reverse transcription polymerase chain reaction (RT-PCR) & Quantitative real time polymerase chain reaction (qRT-PCR) 39
1-9-3 Procedure of RT-PCR 40
CHAPTER TWO MATERIALS AND METHODS 41
2-1 Materials 41
2-1-1 Cultured medium 41
2-1-2 Serum 41
2-1-3 Antibiotics 41
2-1-4 Growth factor 41
2-1-5 PVA-IA film 41
2-1-6 ECM-derived peptides 42
2-1-7 RNA extraction 42
2-1-8 Reverse transcriptase (RT) 42
2-1-9 Real-time PCR (qPCR) 43
2-1-10 Probes for qPCR 43
2-2 Methods and Analysis 43
2-2-1 PVA-IA film preparation 43
2-2-2 Preparation of PVA-IA surfaces grafted with ECM-derived oligopeptides 45
2-2-3 Elasticity measurement of PVA-IA films 46
2-2-4 XPS analysis of dish surface 46
2-2-5 Phosphate buffer saline (PBS) preparation 46
2-2-6 Preparation of FGF-2(b-FGF) protein stock solution 47
2-2-7 Preparation of cell culture medium 47
2-2-8 Cell cultivation 47
2-2-9 Cell density measurements 48
2-2-10 Isolation of total RNA 49
2-2-11 Reverse Transcription of mRNA into cDNA 50
2-2-12 Quantitative real time polymerase chain reaction 51
2-2-13 Imunofluorescence assay 53
CHAPTER THREE RESULTS & DISCUSSION 55
3-1 The elasticity of the PVA-IA films 55
3-2 The XPS measurements of the optimal activation conditions on PVA-IA films 58
3-3 Proliferation of AFSCs cultured on PVA-IA films grafted with ECM-derived oligopeptides having different elasticity 71
3-4 Pluripotency of AFSCs cultured on PVA-IA and PVA-IA films grafted with ECM-derived oligopeptides having different elasticity 84
3-5 Directing AFSCs fates on PVA-IA films by controlling matrix elasticity and extracellular adhesion ligand 91
3-6 The correlation of pluripotency and differentiation in AFSCs cultured on PVA-IA and PVA-IA films grafted with ECM-derived oligopeptides having different elasticity 98
3-7 The expression of pluripotency and differentiation proteins in AFSCs cultured on PVA-IA and PVA-IA films grafted with ECM-derived peptides having different elasticity 104
CHAPTER FOUR CONCLUSIONS 110
CHAPTER FIVE REFERENCES 112

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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2015-7-15
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