博碩士論文 973204008 詳細資訊




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姓名 黃曉茜(Shiau-Chian Huang)  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 微環境對羊水間葉幹細胞多功能性基因表現及分化之影響
(Effect of Micro-environment on Pluripotent Gene Expression and Differentiation of Mesenchymal Stem Cell from Second-trimester Amniotic Fluid)
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摘要(中) 間葉幹細胞在臨床應用上為最方便取得且可自我細胞移植的幹細胞來源之ㄧ,但間葉幹細胞其分化能力不如胚胎幹細胞。近年來有許多研究發現從懷孕婦女的羊水中純化出間葉幹細胞,而純化出的間葉幹細胞為多功能幹細胞並具有像胚胎幹細胞能力可分化三種胚層的細胞。由於微環境 (例如:培養液和培養皿接枝不同官能基或細胞外間質蛋白質) 在間葉幹細胞的基因表現與分化能力有極大的影響,我們將懷孕15至25周的羊水培養於不同的表面材質中,例如電漿改質接上不同的官能基、塗佈或者化學鍵結固定細胞外間質蛋白質於培養皿進行純化與培養繼代,測試多功能性基因表現量與分化能力。我們發現將羊水培養於特定材質的培養皿以純化間葉幹細胞,如纖維蛋白質固定化於改質PS培養皿可得較高的多功能性基因表現,即使經由繼代依舊可維持其多功能性表現;另外,從羊水中純化出的間葉幹細胞除了可分化屬中胚層的脂肪細胞和成骨細胞以外,亦可分化屬外胚層的神經細胞,故從羊水純化之間葉幹細胞為一種多功能幹細胞,並受培養微環境而影響其多功能性基因表現及分化能力。
摘要(英) The cells derived from second-trimester amniocentesis contain pluripotent stem cells capable of differentiating into multiple lineages, including representatives of all three embryonic germ layers. Therefore, amniotic fluid may be a suitable alternative source of damaged cells in our body. Because micro-environment of stem cells (i.e., surface chemistry and topography of culture materials) plays an important role on gene expression and differentiation of stem cells, we isolated stem cells from second-trimester amniotic fluid, and cultured amniotic-derived stem cells on the surface of culture dishes immobilized several ECM proteins by coating or chemical grafting method to generate the nano-segments on the surface (e.g., gelatin, collagen, fibronectin, laminin, vitronectin, Matrigel, and synthetic polymer nano-segments having NH2 group). The culture dishes having those nano-segments were prepared by plasma-polymerization method of glycidylmethacrylate on polystyrene dishes followed by the reaction between epoxy group and ammonia to introduce NH2 groups. Immobilization of proteins on the culture dishes having NH2 groups were performed by using succinimide chemistry. The effect of interaction between amniotic-derived stem cells and nano-segments on the expression and maintenance of pluripotent genes (Oct-4, Sox-2 and Rex-1) was investigated from qRT-PCR and RT-PCR analysis. The amniotic-derived stem cells cultured on the surface of culture dishes having specific nano-segments (e.g., fibronectin) by using chemical grafting method kept pluripotent gene expression of stem cells for a long time. The differentiation ability of amniotic-derived stem cells to osteogenic, adipogenic, and neural cell lineages was also investigated. Differentiation ability to each cell lineages was also dependent on the surface chemistry of culture dishes having specific nano-segments. In conclusion, gene expression of pluripotency and specific differentiation are found to depend on the surface chemistry of culture materials having specific nano-segments.
關鍵字(中) ★ 細胞外間質
★ 多功能基因表現
★ 間葉幹細胞
關鍵字(英) ★ MSC
★ ECM
★ Pluripotent gene expression
論文目次 CHAPTER ONE INTRODUCTION ..................................................................................... 1
1-1 Stem Cell ...................................................................................................................... 1
1-1.1 Potency of stem cells................................................................................................. 1
1-1.1.1 Totipotency ........................................................................................................ 1
1-1.1.2 Pluripotency ...................................................................................................... 1
1-1.1.3 Multipotency ..................................................................................................... 2
1-1.2 Sources of stem cells ................................................................................................. 2
1-1.2.1 Embryonic stem cells (ESCs) ........................................................................... 2
1-1.2.2 Hematopoietic stem cells (HSCs) ..................................................................... 3
1-1.2.3 Mesenchymal stem cells (MSCs) ...................................................................... 4
1-2 Amniotic Fluid Cells ................................................................................................... 8
1-2.1 Amniotic fluid cell type ............................................................................................ 8
1-2.1.1 F-type colonies .................................................................................................. 9
1-2.1.2 AF-type colonies ............................................................................................... 9
1-2.1.3 E-type colonies ................................................................................................ 10
1-2.2 Isolation of amniotic fluid stem cell ....................................................................... 10
1-2.3 Characterization of amniotic fluid stem cell ........................................................... 10
1-2.4 Pluripotency of amniotic fluid stem cells ............................................................... 12
1-3 Effect of micro-environment of mesenchymal stem cells ...................................... 15
1-3.1 Culture medium effect of mesenchymal stem cell .................................................. 16
1-3.2 Effect of culture material for differentiation of mesenchymal stem cell ................ 17
1-3.2.1 Introduction of natural polymeric materials.................................................... 17
1-3.2.2 Conventional synthetic and natural polymeric materials ................................ 21
1-3.2.3 Materials manufactured with nanotechnology and having nanosegments...... 22
1-3.2.4 Application of mesnchymal stem cells ........................................................... 22
1-4 Analysis ...................................................................................................................... 24
1-4.1 X-ray photoelectron spectroscopy .......................................................................... 24
1-4.2 Flow cytometry analysis ......................................................................................... 26
1-4.3 Polymerase chain reaction (PCR) ........................................................................... 26
1-4.3.1 Procedure of PCR............................................................................................ 26
1-4.3.2 Reverse transcription polymerase chain reaction (RT-PCR) and Quantitative real
time polymerase chain reaction (qRT-PCR) ................................................................... 28
1-4.3.3 Procedure of RT-PCR ..................................................................................... 29
CHAPTER TWO MATERIALS AND METHODS ............................................................ 30
2-1 Materials .................................................................................................................... 30
2-2 Methods ...................................................................................................................... 34
2-2.1 Preparation of Cell isolation and culture................................................................. 34
2-2.2 Preparation of culture materials .............................................................................. 35
2-2.3 Analysis ................................................................................................................... 39
3-1 Analysis of materials ................................................................................................. 45
3-2 Morphology of amniotic fluid stem cells cultured on different culture dishes .... 47
3-3 Purification of stem cell from amniotic fluid .......................................................... 54
3-4 Effect of pluripotency gene expression on materials of culture dishes ................ 58
3-5 Effect of differentiation ability of AFCs on materials of culture dishes ............... 63
3-6 Summary .................................................................................................................... 89
CHAPTER FOUR CONCLUSION .................................................................................... 90
CHAPTER FIVE REFERENCES ...................................................................................... 92
CHAPTER SIX Appendix .................................................................................................. 103
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2010-7-15
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