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研究生:許佳如
研究生(外文):Chia-Ju Hsu
論文名稱:羊水間葉幹細胞培養於細胞外間質及材料硬度/彈性表面,其分化能力及多能性之研究
論文名稱(外文):Pluripotency and Differentiation of Stem Cells Cultured on Biomaterials Grafted with Extracellular Matrix Having Different Elasticity
指導教授:樋口亞绀
指導教授(外文):Akon Higuchi
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程學系
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:2013
畢業學年度:101
語文別:英文
論文頁數:89
中文關鍵詞:羊水幹細胞基質軟硬多功能分化能力
外文關鍵詞:amniotic fluid stem cellselasticity of cell culture matrixpluripotencydifferentiation ability
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間葉幹細胞 (MSCs) 是一種在臨床上被廣泛利用的自體幹細胞來源。從羊水中分離出來的幹細胞能夠分化成多種不同的細胞譜系,同時沒有道德上的問題。幹細胞的分化能力以及幹細胞的多能性不只取決於細胞本身,同時也會受到外在環境的影響。因此,使用天然的生物分子 (如細胞外間質) 模仿細胞的微環境,增加幹細胞在體外生長的數量以及調控幹細胞的分化對再生醫學是一件非常重要的研究。在這篇研究中,我們將羊水幹細胞(AFSCs)分別培養在不同軟硬度PVA-IA膜且有接枝collagen或fibronectin等細胞外間質 (ECM) 的膜上,以分析羊水幹細胞與細胞外間質的交互作用。可透過交聯時間(0.5小時-48小時)控制PVA-IA膜的軟硬度(3.7千帕-30.4千帕)。相對於AFSCs培養於較硬的膜上,AFSCs培養在較軟的膜上有較小的延展性、應力纖維和較慢的增殖率。測試AFSCs培養在含有接枝ECM不同軟硬度膜間的多能性基因表現。結果顯示,AFSCs培養在軟的膜(10.3千帕)有較好的多能性基因表現,也代表在此條件下可以維持AFSCs多功能,亦可拉長期培養時間。同時,也測試當細胞培養在不同軟硬度的膜上,基質對AFSCs的分化結果。結果發現,AFSCs分別培養在10.3千帕、19.6千帕、30.4千帕時,AFSCs會分化成神經細胞(Nestin)、平滑肌細胞(巢)(α-actin)、骨細胞(Runx2)。

Mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Stem cells derived from amniotic fluid (AFSCs) are pluripotent fetal cells capable of differentiating into multiple lineages, containing representatives of all the three embryonic germ layers. AFSCs are categorized as the intermediate stage between the embryonic stem cells (ESCs) and adult stem cells, and AFSCs may have a distinct mechanism to choose their fate. Therefore, amniotic fluid represents a rich and more suitable source of stem cells in regenerative medicine and tissue engineering than ESCs and induced pluripotent stem cells (iPSCs) due to the lack of ethical concerns regarding use of ESCs and the lack of concerns about xenogenic contamination arising from the use of mouse embryonic fibroblasts as a feeder layer for iPSCs and ESCs. However, stem cells fate of decision is regulated not only by stem cells themselves, but also the effect of microenvironment of stem cells. Therefore, stem cell microenvironment surrounding specific matrix proteins should be important to decide the stem cell fate of differentiation. The optimal design of stem cell culture biomaterials will facilitate the in-vitro production of the large numbers of pluripotent stem cells and specifically more numbers of differentiated cells, which are demanded in the regenerative medicine.
In this study, we investigated whether the stiffness of cell adhesion substrates affect the maintenance of pluripotency of the stem cells and/or modulated the stem cell fate of differentiation. Stem cells from amniotic fluid were cultured on different stiffness of polyvinylalcohol-co-itaconic acid (PVA-IA) films grafted with extracellular matrix (ECM) dishes where collagen, and fibronectin were selected for further studies as ECM components (nanosegments). PVA-IA films were selected as the base matrix due to easy regulation of elasticity (3.7 kPa- 30.4 kPa) by changing the crosslinking time (0.5 hr – 48 hr). AFSCs on soft substrates (3.7 kPa-10.6 kPa) had less spreading, fewer stress fibers and lower proliferation rate than AFSCs on stiff substrate (25.3 kPa-30.4 kPa). The effects of interaction between AFSCs and ECMs(nanosegments) were investigated on the expression of pluripotent genes (e.g., Nanog, Sox2, and Oct4 ) of AFSCs and on the differentiation abilities of AFSCs into several lineage, which were cultured on PVA-IA films grafted with collagen, and fibronectin having different elasticity at passage 4. It was found that AFSCs on PVA-IA films grafted with ECM of elastic modulus (10.3 kPa-12.2 kPa) in the soft showed higher pluripotency genes than hard substrates. AFSCs could differentiate into neural (nestin), smooth muscle cell (α-actin), or osteogenic (Runx2) phenotypes depending on whether they were cultured on PVA-IA films grafted with ECM substrates of elastic modulus in the lower, intermediate or higher ranges. AFSCs cultured on soft substrates have weaker cell adhesion, and showed higher expression of early marker of neural cells (nestin) in expansion medium without addition of induction molecules.

CHAPTER ONE INTRODUCTION 1
1-1 Mesenchymal stem cells 1
1-2 Amniotic fluid-derived stem cells 3
1-2-1 Amniotic fluid cells 3
1-2-2 Amniotic fluid cell types 4
1-2-3 Isolation of amniotic fluid stem cells (AFSCs) 7
1-2-4 Characterization of amniotic fluid stem cells 8
1-2-5 Pluripotency of amniotic fluid stem cells 10
1-3 Microenvironment of stem cells 12
1-3-1 Soluble factors 13
1-3-2 Cell-cell interactions 13
1-3-3 Cell-biomaterials interactions 13
1-3-4 Physical factors 14
1-4 Type and classification of artificial ECMs 16
1-5 Markers of piuripotent gene 21
1-6 Markers of differentiation lineages of MSCs 23
1-7 Polymerase chain reaction (PCR) 25
1-7-1 Procedure of PCR 26
1-7-2 Reverse transcription polymerase chain reaction (RT-PCR) &Quantitative real time polymerase chain reaction (qRT-PCR) 28
1-7-3 Procedure of RT-PCR 29
CHAPTER TWO MATERIALS AND METHOD 30
2-1 Materials 30
2-1-1 PVA-IA film 30
2-1-2 Cultured medium 30
2-1-3 Serum 30
2-1-4 Antibiotic 31
2-1-5 Growth factor 31
2-1-6 ECM proteins 31
2-1-7 RNA extraction 31
2-1-8 Reverce transcriptase (RT) 31
2-1-9 Real - time (qPCR) 31
2-1-10 Probes 32
2-2 Method and Analysis 32
2-2-1 PVA-IA film preparation 32
2-2-2 Preparation of PVA-IA coating dish grafted with ECM 33
2-2-3 Elasticity measurement of PVA-IA 34
2-2-4 XPS analysis of dish surface 36
2-2-5 Phosphate buffer saline (PBS) preparation 36
2-2-6 Preparation of FGF-2 (b-FGF) protein stock solution 36
2-2-7 Preparation of cell cultured medium 37
2-2-8 Cell cultivation 37
2-2-9 Cell density measurement 38
2-2-10 Isolation of total RNA 39
2-2-11 Reverse Transcription of mRNA into cDNA 40
2-2-12 Quantitative real time polymerase chain reaction 41
CHAPTER THREE RESULTS & DISCUSSION 43
3-1 The elasticity of the PVA-IA films 43
3-2 The XPS measurements of the PVA-IA and ECM-grafted PVA-IA films having different elasticity 44
3-3 Morphology of AFSCs cultured on PVA-IA and ECM-grated PVA-IA films having different elasticity 60
3-4 Pluripotency of AFSCs cultured on PVA-IA and ECM-grafted PVA-IA films having different elasticity 65
3-5 PVA-IA and ECM-grafted PVA-IA films having different elasticity guide AFSCs into specific lineages of differentiation 72
CHAPTER FOUR CONCLUSION 80
CHAPTER FIVE REFERENCE 82

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