博碩士論文 993204066 詳細資訊




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姓名 蕭瑞祥(Jui-hsiang Hsiao)  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 於不同彈性係數的生醫材料上體外培植造血幹細胞
(Ex Vivo Expansion of Hematopoietic Stem Cells Cultured on Biomaterial Having Different Elasticity)
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摘要(中) 細胞培植基材表面的彈性在幹細胞的分化上扮演著重要的角色,不論是培養基材的軟硬程度或是彈性係數(物理特性)在影響幹細胞的命運上都是具有決定性的因素。同樣的,在二維的平面上培植幹細胞,培植基材表面的化學特性與生物特性也都會對幹細胞產生影響。在本研究中發現,在表面不具有任何生物特性改質的polyvinylalcohol-co-itaconic acid (PVA-IA) 薄膜上體外培植造血幹細胞,於表面基材彈性係數30.4kPa 的條件下最適合造血幹細胞體外培養。但細胞培植基材(PVA-IA) 薄膜在經過改質表面具有細胞外基質或是寡肽之後 (生物特性訊號),實驗得到的趨勢以及結果卻大不相同。造血幹細胞培養於彈性係數12.2kPa介在中間值的培養基材上有著最高的體外增質倍率。此一研究結果顯示: 不論是物理特性還是生物特性都將對造血幹細胞體外增質造成影響,而且生物特性在二維平面的體外培植上似乎扮演著較重要的角色。除此之外,此一研究結果也顯示: 在不同的生物特性及物理特性條件下,幹細胞的是否分化及其分化的途徑也會有所差異。
摘要(英) Matrix elasticity play an important role in stem cell lineage specification in differentiating either stiffness or elasticity (physical property) of the culture substrates is one of the selective causes for affects the fate determination of stem cell. Similarly, the chemical and biological properties of matrix can also affect the stem cell of various functions on 2D cultivate surface. In the present investigation, it was found that the stiffer surface with elasticity of 30.4 kPa or 16.5 MPa was optimal for the ex vivo expansion of HSCs without any further biological modification on polyvinylalcohol-co-itaconic acid (PVA-IA) films. On the contrary, it came up with different results after grafting the extracellular matrix or oligopeptide contributing with HSC binding domain. The intermediate stiffness surface with elasticity of 12.2 kPa or 1.417 MPa had the highest expansion fold of HSCs. This result indicated that both physical and biological properties would affect the ex vivo expansion of HSCs. The biological cues seem to play a more significant role during ex vivo expansion of HSCs on the 2D cultivation. Further findings suggested that the effect of optimal biological and physical parameters required for the different lineages of various stem cells.
關鍵字(中) ★ 造血幹細胞
★ 彈性
★ 細胞外基質
★ 臍帶血
關鍵字(英) ★ Hematopoietic stem cell
★ Elasticity
★ Extracellular Matrix
★ Umbillical Cord Blood
論文目次 Index of Content
Introduction...................................1
1.1 Stem cell................................. 1
1.2 Stem cell therapy......................... 2
.1 Hematopoietic Stem Cell.......................................... 3
1-3.2 HSC micro niche: Bone marrow environment................................... 4
1-4 Cytokines and cellular mechanisms of hematopoiesis................................. 5
1-5 Purification methods of HSCs.......................................... 9
1-5-1 Fluorescence-activated cell sorting (FACS)....................................... 10
1-5-2 Magnetic-activated cell sorting (MACS) ............................................. 12
1-6 Extracellular matrix (ECM) and nano-segment...................................... 15
1-6-1 Type and classification of artificial ECMs......................................... 16
1-6-1.1 Collagen............................. 19
1-6-1.2 Fibronectin.......................... 19
1-6-1.3 Laminin ............................................. 20
1-6-1.4 Vitronectin.......................... 20
1-6-1.5 Matrigel............................. 21
1-6-2 The effect of extracellular matrix (ECM) to stem cells........................................ 22
1-7 Physical cues affect ex vivo expansion ............................................. 23
1-7.1Physical cue affects ex vivo expansion of stem cells........................................ 25
1-8Ex vivoexpansion of hematopoietic stem cells........................................ 27
1-8.1Culture medium of ex vivoexpansion of HSCs......................................... 30
1-9Conventional synthetic and natural polymeric materials.................................... 30
1-9-1 Effect of surface chemistry on the ex vivo expansionof HSC.......................................... 35
1-9-2Polymeric materials modified with biological cues ............................................. 36
1-10 HSC analysis ............................................. 44
1-10-1Flow cytometry analysis..................................... 44
1-10-2 Colony Forming cell (CFC) assay........................................ 47
Method and Material...........................49
2-1 Material ................................ 49
2-2 PVA-IA film Preparation.................................. 49
2-3 Preparation of PVA-IA coating dish grafted with ECM and oligopeptide ............... 50
2-4Elasticity measurement of PVA-IA ............................................. 51
2-4.1 Dynamic mechanical analysis..................................... 51
................................................................................................................................ 512-4.2 Atomic Force Microscope................................... 52
2-5 Thickness measurement via scanning electric microscope ............................................. 54
2-6 XPS analysis of dish surface ............................................. 54
2-7 Material for ex vivo expansion of hematopoietic stem cells........................................ 54
2-8 Buffer Solution.......................... 55
2-9 Sterilization of Modified Materials.................................... 55
2-10 Purificationof hematopoietic stem cells ............................................. 56
2-10-1 Magnetic labeling and magnetic separationof CD34+
cells ....................................... 57
2-11 Ex vivo Expansion of HSCs on PVA-IA coatedDishes (2D Culture)..................................... 60
2-12 HSCs Analysis........................... 61
2-12.1 Flow cytometry analysis..................................... 61
2-12.2 Colony Forming cell (CFC) assay........................................ 61
2-12.3 Immunofluorescence ............................................. 62
Result and Discussion ..............................................64
3-1 Preparation of PVA-IA with different stiffness ............................................. 64
3-2 The scale effect of PVA-IAfilm....................................... 64
3-3 The thickness of the PVA-IAcoated on TCPS......................................... 68
3-4 The elasticity of the PVA-IAgel........................................ 71
3-5 The XPS measurement of the PVA-IA films........................................ 78
3-6 Ex vivo expansion of HSC from UCB on PVA-IA films having different stiffness. 82
3-7 Colony forming assay of HSC from UCB on variety stiffness dishes............... 87
3-8 Ex vivo expansion of HSC from UCB on PVA-IA films having biological cues and
different stiffness ............................................. 92
3-9 Colony forming assay of HSC from UCB on variety stiffness dishes with biological
cues......................................... 96
Conclusion ..................................100
Reference….……,,……………………………….....…………………………………103
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指導教授 樋口亞紺(Higuchi Akon) 審核日期 2013-7-8
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