博碩士論文 993204033 詳細資訊




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姓名 趙俊凱(Chun-Kai Chao)  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細胞外間質改質表面其分化能力及多能性之研究
(DIFFERENTIATION ABILITY AND PLURIPOTENCY OF AMNIOTIC FLUID-DERIVED STEM CELLS CULTURED ON EXTRACELLULAR MATRIX-IMMOBILIZED SURFACE)
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摘要(中) 胚胎幹細胞 (ESCs) 在使用時常會遇到道德上的問題,而誘導型多能性幹細胞 (iPSCs) 在培養時常會受到飼養層的汙染。間葉幹細胞 (MSCs) 是一種在臨床上最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。因此,羊水幹細胞比胚胎幹細胞 (ESCs) 和誘導型多能性幹細胞 (iPSCs) 更適合應用在組織工程以及再生醫學。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,我們將羊水幹細胞分別培養在接枝有 gelatin, collagen, fibronectin, laminin, vitronectin和Matrigel 等細胞外間質 (ECM) 的培養皿上,以分析羊水幹細胞與細胞外間質的交互作用。接枝有細胞外間質的培養皿水接觸角大約在40到65度之間,非常適合細胞生長。羊水幹細胞培養在細胞外間質接枝的培養皿上增強了幹細胞分化為成骨細胞的能力。此發現說明了羊水幹細胞在與細胞外間質的交互作用下有助於幹細胞分化為成骨細胞。
在培養皿上改植適合的奈米片段 (細胞外間質) 能夠促進羊水幹細胞分化為成骨細胞。奈米片段的選擇取決於希望幹細胞分化成何種譜系,我們將會討論幹細胞在長時間培養繼代後,哪種細胞外間質能夠維持羊水幹細胞的多能性以及羊水幹細胞的分化能力。
摘要(英) Mesenchymal stem cells (MSCs) are one of the most widely available autologous sources of stem cells for clinical applications. Stem cells derived from amniotic fluid are pluripotent fetal cells capable of differentiating into multiple lineages, including representatives of the three embryonic germ layers. Therefore, amniotic fluid may become a more suitable source of stem cells in regenerative medicine and tissue engineering than embryonic stem cells (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. 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. Therefore, mimicking stem cell microenvironments using natural biomacromolecules, such as extracellular matrix (ECM) proteins, facilitates the in vitro production of the large numbers of pluripotent stem cells and specifically differentiated cells needed for regenerative medicine
In this study, stem cells from amniotic fluid were cultured for several passages on dishes grafted with extracellular matrix (ECM) or Matrigel where gelatin, collagen, fibronectin, laminin, and vitronectin were selected as ECM components (nanosegments). The effects of interactions between amniotic fluid stem cells and nanosegments were investigated on the expression of pluripotent genes (e.g., Oct4 and Nanog) and on the differentiation abilities of osteoblasts at each passage. The ECM-grafted dishes produced water contact angles from 40 to 65 degrees, which was an adequate water contact angle range for the cell culture. Culture on ECM-immobilized dishes enhances amniotic fluid stem cell differentiation into osteoblasts more than culture on polystyrene dishes grafted with amino groups (PS-NH2 dishes). This finding indicates that specific interactions between amniotic fluid cells and the ECM grafted onto the culture dishes promote the differentiation of cells into osteoblasts. Immobilization of the optimal nanosegments (ECM or Matrigel) onto culture dishes enhances amniotic fluid stem cell differentiation into osteoblasts; the choice of nanosegments depends on the desired differentiated cell type. We will discuss the optimal ECM-grafted dishes, which keep pluripotency of the amniotic fluid stem cells for a long time (i.e., at late passages).
關鍵字(中) ★ 成骨分化
★ 多能性
★ 羊水幹細胞
★ 細胞外間質
關鍵字(英) ★ pluripotency
★ extracellular matrix
★ amniotic fluid stem cells
★ osteogenic differentiation
論文目次 CHAPTER ONE INTRODUCTION 1
1-1 MESENCHYMAL STEM CELLS 1
1-2 AMNIOTIC FLUID-DERIVED STEM CELLS 4
1-2-1 Amniotic fluid cells 4
1-2-2 Amniotic fluid cell types 4
i. F-type colonies 4
ii. AF-type colonies 6
iii. E-type colonies 6
1-3 Isolation of amniotic fluid stem cells (AFSCs) 7
1-4 Characterization of amniotic fluid stem cells 8
1-5 Pluripotency of amniotic fluid stem cells 10
1-6 MICROENVIRONMENT OF STEM CELLS 13
1-6-1 Soluble factors 14
1-6-2 Cell-Cell interactions 14
1-6-3 Cell-biomaterials interactions 14
1-6-4 Physical factors 15
1-7 EXTRACELLULAR-MATRICES 16
1-8 OSTEOGENIC DIFFERENTIATION 20
1-8-1 The process of bone development in situ 20
1-8-2 Developmental pathways for bone formation 21
1-8-3 ECM substratum for promoting osteogenic differentiation 25
1-8-4 The markers of osteogenic differentiation 26
CHAPTER TWO MATERIALS AND METHODS 28
2-1 MATERIALS 28
2-1-1 Culture medium 28
2-1-2 Differentiation medium 28
2-1-3 Serum 28
2-1-4 Antibiotics 28
2-1-5 Bovine serum albumin (BSA) 28
2-1-6 Growth factor 29
2-1-7 ECM proteins 29
2-1-8 RNA extraction 29
2-1-9 Reverse transcriptase (RT) 30
2-1-10 Polymerase chain reaction (PCR) 30
2-1-11 Primers 30
2-1-12 Real-time PCR 31
2-1-13 Probes 31
2-1-14 Chemical for observation of differentiated cells 31
2-2 METHODS AND ANALYSIS 32
2-2-1 Preparation of phosphate buffer saline (PBS) 32
2-2-2 Preparation of FGF-2 (b-FGF) stock solution 32
2-2-3 Preparation of cell cultured medium 32
2-2-4 Cell cultivation 33
2-2-5 Cell density measurement 33
2-2-6 Cell differentiation 34
2-2-7 Preparation of surface modified polystyrene (PS) dishes 35
2-2-8 Preparation of ECM-immobilized dishes 38
2-2-8-1 Chemical grafting method 38
2-2-8-2 Coating method 38
2-2-9 Preparation of Pluronic grafting dishes 39
2-2-10 Isolation of total RNA 40
2-2-11 Reverse Transcription of mRNA into cDNA 40
2-2-12 Quantitative PCR 42
2-2-13 PCR 43
2-2-14 X-ray photoelectron spectroscopy (XPS) 44
2-2-15 Water contact angle 45
2-2-16 Mineral deposition by Alizarin Red S or von Kossa staining 45
2-2-17 Measurement of alkaline phosphatase (ALP) activity 46
CHPTER THREE RESULTS AND DISCUSSION 48
3-1 CHARACTERIZATION OF IMMOBILIZED-DISHES 48
3-2 MORPHOLOGY OF AFSCS CULTURED ON DIFFERENT ECM-IMMOBILIZED DISHES 61
3-3 PLURIPOTENCY OF AFSCS CULTURED ON DIFFERENT GRAFTED DISHES 67
3-4 OSTEOGENIC DIFFERENTIATION OF AFSCS CULTURED ON GRAFTED DISHES 71
3-5 THE RELATIONSHIP BETWEEN DOUBLING TIME AND PLURIPOTENCY OF AFSCS 95
3-6 THE RELATIONSHIP BETWEEN DIFFERENTIATION AND PLURIPOTENCY OF AFSCS 98
CHAPTER FOUR CONCLUSION 101
SUPPLEMENTARY DATA 103
CHAPTER FIVE REFERENCE 108
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2012-7-9
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