博碩士論文 983204026 詳細資訊




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姓名 陳立穎(Li-Ying Chen)  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 利用具有奈米片段與細胞外間質蛋白質的表面改殖材質進行臍帶血造血幹細胞體外培養
(Ex Vivo expansion of Hematopoietic Stem Cells on Surface-modified Materials having Nano-segments and Extracellular Matrix Proteins from Umbilical Cord Blood)
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摘要(中) 臍帶血是造血幹細胞移殖可行的幹細胞來源。然而從單一臍帶血中能取得的造血幹細胞數量極低,限制了對病患的臍帶血移殖療。
在本研究中,我們使用兩種不同的培養方式,各別是二維平面與三維立體培養法。對2維平面培養,我們使用具有細胞外間質(ECM)與寡糖胜肽蛋白質(如Fibronectin與CS-1)或者是化學官能基(如氨基)固定化接枝的的表面改殖材料進行培養。除此之外,在三維立體培養法上,我們也發展了使用膜過濾法的造血幹細胞直接體外培養法;我們研究如何使用奈米接枝對材料或膜表面改殖,並模仿骨髓幹細胞龕環境,以適用於體外造血幹細胞培養。
對直接體外培養法而言,使用具有COOH基接枝的PU膜具有較高的幹細胞增殖倍率;對膜過濾直接體外培養法而言,潤洗液必需含有超過50%的低血小板血清。使用膜過濾直接體外培養法可藉由簡單的臍帶血分離操作提供三維培養環境與高幹細胞增殖倍率。在二維培養方面,高表面接枝密度氨基不適合培養造血幹細胞,我們發現對造血幹細胞培養而言,氨基具有最適化表面接枝密度。除此之外,在最適化接枝密度下且具有良好表面接枝度控制的細胞外間質表面改殖,有較高的幹細胞增殖倍率。總結而言,PS-FN、PS-CS1-H與PS-Ozone-AMA-0.3mM是出色的造血幹細胞培養生醫材料。
摘要(英) Umbilical cord blood (UCB) is a promising source of stem cells for hematopoietic stem cell (HSC) transplantation. However, the low number of HSCs that can be obtained from a single UCB donor limits the transplantation of UCB for patients. We investigated two culture methods of HSCs, 2D and 3D culture. The 2D-culture used the surface-modified materials immobilized extracellular matrix (ECM) and oligopeptides such as fibronectin and CS-1, or nano-segments having amino group. We also developed a direct ex vivo HSC expansion method followed by 3D-culture of HSCs on the membranes. We investigated how to create and mimic the bone marrow niche for the expansion of HSCs by using nano-segments and ECMs on the surface of the membranes and culture materials. In the direct ex vivo expansion of HSCs, PU membranes having nano-segments of carboxylic acid showed higher expansion fold of HSCs among surface-modified PU membranes investigated in this study. In this case, the rinsing solution should contain platelet-poor plasma (PPP) more than 50%. The direct ex vivo expansion of HSCs from UCB filtered through membranes can provide a simple 3-D culture method of HSCs and a high fold ex vivo expansion of HSCs with a simple operation. In 2D-culture of HSCs, high surface density of amino group on the culture dishes was not favor, and the optimal surface density of amino group was found for HSC expansion. There found optimal amino groups with well controlled surface density and ECMs (CS1-H and Fibronectin) with optimal surface density for high expansion folds of HSCs in 2-D culture. In summary, PS-FN, PS-CS1-H and PS-Ozone-AMA-0.3mM dishes are excellent cultivation materials of HSCs.
關鍵字(中) ★ 臍帶血
★ 造血幹細胞
★ 生醫材料
★ 體外培養
★ 幹細胞
關鍵字(英) ★ Umbilical Cord Blood
★ Extracellular Matrix Proteins
★ Nano-segament
★ Ex Vivo expansion
★ Hematopoietic Stem Cells
論文目次 INDEX OF CONTENTS
摘要 iv
ABSTRACT v
致謝 vi
ACKNOWLEDGEMENT vii
INDEX OF CONTENTS viii
INDEX OF FIGURES xi
INDEX OF TABLES xv
CHAPTER ONE: INTRODUCTION 1
1-1 Stem Cell 1
1-1-1 Sources of Stem Cells 2
1-2 Hematopoietic Stem Cells 3
1-2-1 Types of HSCs 3
1-2-2 Cytokines and Cellular Mechanisms of Hematopoiesis 4
1-3 Purification Methods of HSCs 7
1-3-1 Fluorescence-Activated Cell Sorting (FACS) 7
1-3-2 Magnetic-Activated Cell Sorting (MACS) 10
1-3-3 Membrane Purification Method 11
1-4 Extracellular Matrix (ECM) and Nano-segment 12
1-4-1 Type and Classification of Artificial ECMs 13
1-4-1.1 Collagen 16
1-4-1.2 Fibronectin 17
1-4-1.3 Laminin 18
1-4-1.4 Vitronectin 18
1-4-1.5 Matrigel 18
1-4-2 The Effect of Extracellular Matrix (ECM) to Stem Cells 19
1-5 Ex Vivo Expansion of Hematopoietic Stem Cells 20
1-5-1 Culture Medium of Ex Vivo Expansion of HSCs 22
1-6 Culture Materials for Ex Vivo Expansion of HSCs 23
1-6-1 Conventional Synthetic and Natural Polymeric Materials 24
1-6-2 Materials Modification with Nanotechnology 29
1-6-3 Plasma-surface Modification of Biomaterials 34
1-6-4 Polymeric Materials Modified with Immobilized Proteins and Oligopeptides 35
1-6-5 Polymeric Materials Modified With Immobilized Glycosaminoglycans 40
1-6-6 Materials for 3D culture 43
1-7 HSCs Analysis 45
1-7-1 Flow Cytometry Analysis 45
1-7-1-1 The Use of the CD34 Surface Marker to Characterize Primitive Cells 46
1-7-1-2 Flow Cytometry Protocols For the Enumeration of CD34+ cells 46
1-7-2 Colony Forming Cell (CFC) Assay 48
CHAPTER TWO: MTERIALS AND METHOD 50
2-1 Materials 50
2-1-1 Preparation of Surface-Modified PU Foaming Membranes and Surface-Modified Polystyrene Dishes 51
2-1-2 Preparation of PS-NH2 Dishes Immobilized With Extracellular Proteins and Peptides. 54
2-1-3 Preparation of PS-AMA (2-Aminoethyl methacrylate hydrochloride) surface Dishes 55
2-1-4 Preparation of Platelet Poor Plasma (Plasma A) 57
2-1-5 Buffer Solution 58
2-1-6 Sterilization of Modified Materials 58
2-2 Characterization Analysis 59
2-2-1 Scanning Electron Microscopy (SEM) analysis 59
2-2-2 X-ray Photoelectron Spectra (XPS) 59
2-2-3 Water Contact Angle 60
2-3 HSCs Purification 60
2-3-1 Membrane Purification method 60
2-3-2 Magnetic Cell Sorting (MACS) method 62
2-3-2-1 Preparation of Cord Blood Cells 62
2-3-2-2 Magnetic Labeling and Magnetic Separation of CD34+ Cells 63
2-4 Ex Vivo Expansion of HSCs 66
2-4-1 Direct Ex Vivo Expansion of HSCs on Membranes 66
2-4-2 Ex vivo Expansion of HSCs on Surface-modified Dishes (2D Culture) 66
2-5 HSCs Analysis 67
2-5-1 Flow Cytometry Analysis 67
2-5-2 Colony Forming Cell (CFC) Assay 68
CHAPTER THREE: RESULTS AND DISCUSSION 70
3-1 Characterization of the Surface of Modified Dishes 70
3-2 Direct Ex Vivo Expansion of HSCs from UCB by Membrane Filtration Method 79
3-2-1 Effect of Recovery Solution Using Different Diluted Ratio of PPP Permeated through PU-COOH Membranes Having Pore Size = 11 μm 79
3-2-2 Direct Ex vivo Expansion of HSCs on Membranes 83
3-3 Ex Vivo Expansion of HSCs from UCB on Surface-modified Dishes with 2D Cultivation 89
3-3-1 Ex Vivo Expansion of HSCs from UCB on AMA Modified Dishes 92
3-3-1-1 Ex Vivo Expansion of HSCs from UCB on PS-ATRP-AMA Modified Dishes 92
3-3-1-2 Ex Vivo Expansion of HSCs from UCB on PS-Ozone-treatment-AMA Modified Dishes 102
3-3-2 Ex Vivo Expansion of HSCs from UCB on ECM- and oligopeptide-immobilized Dishes by Plasma Treatment 109
CHAPTER FOUR: CONCLUSION 123
REFERENCES 126
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2011-6-29
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