博碩士論文 106324013 詳細資訊




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姓名 李雨駿(Yu-Chun Lee)  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 膜遷移法藉由細胞外間質塗覆薄膜從脂肪組織分離出脂肪幹細胞
(A Hybrid-Membrane Migration Method to Isolate Adipose-Derived Stem Cells from Fat Tissues Through Membranes Coated with Extracellular Matrices)
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摘要(中) 人體脂肪幹細胞(hADSCs)可以通過從脂肪組織中分離獲得,且hADSCs目前是比人體誘導多能幹細胞(hiPSC)和胚胎幹細胞(hESC)更實用的幹細胞來源。目前,多數的臨床試驗使用hADSCs,而僅使用hiPSC和hESC進行了少數臨床試驗。已知hADSCs顯示異質性特徵並且包含不同的多能性和分化能力。因此,我們預期通過不同分離方式分離的hADSCs,其幹細胞特徵,多能性和分化能力應該有所不同。普遍利用基質血管部分(SVF,原生hADSCs溶液)的細胞培養來得到hADSCs,其中SVF溶液可通過膠原酶(Collagenase)消化脂肪組織並且離心後獲得。依不同純化方式所分離出的人類脂肪幹細胞會具有不同的純度及多樣性。在實驗室,我將通過網狀尼龍膜過濾器的膜遷移方法進行創新,脂肪組織溶液經由此方法,使其hADSCs純化且具有極高的純度和多能性。其方法將原生脂肪組織溶液通過孔徑為8至25μm的多孔膜(例如:尼龍網),並將膜放入細胞培養液中培養15-18天。
就我們所知,其生長的微環境於人類脂肪幹細胞之基因表現及分化能力中,扮演著重要的角色。因此在此研究中,新的膜遷移方法是使用最佳孔徑(11和20μm)的尼龍網膜和PLGA絲網膜開發的,其中最佳細胞外間質(ECM)塗覆在膜上,此方法可以純化hADSCs。期望利用優化後的純化方法所獲得之脂肪幹細胞能具有較高多能性,以及細胞分化能力,如:軟骨細胞、成骨細胞及脂肪細胞之分化。我們利用膜遷移方法將脂肪組織分離出hADSCs,並且在此方法中使用了不同的膜,如:(a)尼龍網(孔徑為11跟20μm)和PLGA絲網膜、(b)尼龍網(孔徑為11跟20μm)和PLGA絲網膜塗佈第一型膠原蛋白(Collagen type I)、(c)尼龍網(孔徑為11跟20μm)和PLGA絲網膜塗佈人類纖維連接蛋白(Fibronectin)、(d)尼龍網(孔徑為11跟20μm)和PLGA絲網膜塗佈人類重組玻璃粘連蛋白(human recombinant Vitronectin)。第一型膠原蛋白為異種來源的材料,其餘細胞外間質之來源則為人類(無異種材料)。從膜遷移下來的人體脂肪幹細胞(hADSCs)與使用普通培養方法分離的細胞相比較表現極高百分比(例如:> 98%)的間充質幹細胞標記物(CD44,CD73和CD90),並且在一些多能性基因(Oct4,Sox2和Nanog)的表現量高出一個級距。
摘要(英) Human adipose-derived stem cells, hADSCs, can be obtained by isolation from fat tissue, which is currently a more practical source of stem cells than human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Currently, several clinical trials use hADSCs, whereas only a few clinical trials have been performed using hiPSCs and hESCs. However, hADSCs are known to show heterogeneous characteristics and contain different pluripotency and differentiation abilities. Therefore, it is expected that the stem cell characteristics, pluripotency, and differentiation abilities should be different for hADSCs isolated by different isolation methods. hADSCs are typically isolated by cell culture of stromal vascular fraction (SVF, primary hADSC solution) where the SVF solution can be obtained by collagenase digestion of fat tissues followed by centrifugation. The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. It is innovated that the membrane migration method through Nylon mesh filter purifies hADSCs from a fat tissue solution with extremely high purity and pluripotency in my laboratory. A primary fat-tissue solution was permeated through the porous membranes (e.g., Nylon mesh) with a pore size from 8 to 25 μm, and the membranes were incubated in cell culture medium for 15-18 days.
In this study, I developrd a new membrane migration method using Nylon mesh membranes having optimal pore sizes, 11 and 20 μm, and PLGA/silk membranes where optimal extracellular matrix (ECM) was coated on the membranes, which could purify hADSCs. The isolated hADSCs are expected to have high pluripotency and high differentiation ability into chondrocytes, osteoblasts and adipocytes. hADSCs were isolated from adipose tissue by the membrane migration method where different membranes were used, e.g., (a) Nylon mesh and PLGA (poly (lactic-co-glycolic acid))/silk screen membrane, (b) Nylon mesh and PLGA/silk screen membrane coated with collagen type I, (c) Nylon mesh and PLGA/silk screen membrane coated with human recombinant-vitronectin, (d) Nylon mesh and PLGA/silk screen membrane coated with human fibronectin. Collagen type I is xeno-containing materials, whereas another extracellular matrices (ECMs) were xeno-free materials. The hADSCs that migrated from the membranes kept an extremely high percentage (e.g., >98%) expression of mesenchymal stem cell markers (CD44, CD73, and CD90) and showed almost one order of magnitude higher expression of some pluripotency genes (Oct4, Sox2, and Nanog) compared with cells isolated using the conventional culture method.
關鍵字(中) ★ 脂肪幹細胞
★ 細胞外間質
★ 膜分離法
關鍵字(英) ★ Adipose derived stem cell
★ Extracellular matrix
★ membrane migration method
論文目次 Abstract i
Index of Content iii
Index of Figure vii
Index of Table xi
Chapter 1. Introduction 1
1-1 Stem cell therapies in clinical trials 1
1-1.1 Stem cells 2
1-1.2 Pluripotent stem cells (PSCs) 4
1-1.3 Mesenchymal stem cells (MSCs) 5
1-2 Cell migration 7
1-3 Adipose derived-stem cells (ADSCs) 8
1-3.1 Isolation of adipose derived-stem cells 9
1-3.2 Surface immunophenotype marker of ADSCs 10
1-3.3 Purification of membrane filtration and migration method 13
1-3.4 Differentiation abilities of adipose derived-stem cells 14
1-4 Extracellular matrix (ECM) 17
1-4.1 Collagen (Col) 18
1-4.2 Fibronectin (FN) 20
1-4.3 Vitronectin (VN) 21
1-5 Poly (lactic-co-glycolic acid) (PLGA)/silk screen membrane 22
1-6 Goal of this study 23
Chapter 2 Materials and Methods 25
2-1 Experimental materials 25
i. Adipose tissue 25
ii. Common cell cultured dish 26
iii. Isolation process 26
iv. Membrane migration method 26
v. Isolation process 27
vi. Passage process 27
vii. pH standard solution 27
viii. Phosphate buffer saline solution (PBS) 27
ix. Flow cytometry 28
x. RNA extraction kit 28
xi. Reverse transcription kit 28
xii. Real-time polymerization chain reaction 28
xiii. qRT-PCR probe 29
xiv. Osteogenic differentiation 29
xv. Chondrogenic differentiation 30
xvi. Adipogenic differentiation 30
2-2 Experimental methods 31
2-2.1 Preparation of phosphate buffer solution (PBS) 31
2-2.2 Preparation of culture medium 31
2-2.3 Isolation of adipose-derived stem cells (hADSCs) 31
2-2.4 Cultivation and passage of hADSCs 33
2-2.5 Cell density measurement 35
2-2.6 Preparation of PLGA/silk screen membrane 36
2-2.7 Preparation of ECM-coated membranes 37
2-2.8 Pluripotent gene expression analysis 38
2-3.9 Immunofluorescence staining 42
2-3.10 Flow-cytometry measurements 43
2-3.11 Quantitative analysis of differentiation 44
2-3.12 Osteogenic differentiation 45
2-3.13 Alkaline phosphate activity (ALP activity) 45
2-3.14 Alizarin red S staining assay 47
2-3.15 von Kossa staining 47
2-3.16 Adipogenic differentiation of hADSCs 48
2-3.17 Oil Red O staining 48
2-3.18 Chondrogenic differentiation of hADSCs 48
2-3.19 Alcian blue staining 49
Chapter 3 Results and Discussion 50
3-1 Structure of Nylon and PLGA/silk screen membranes 50
3-2 Cultivation of hADSCs 55
3-2.1 Morphology and doubling time of passage 5 hADSCs after membrane migration method 56
3-2.2 The cell ratio of hADSCs (passage 5) by membrane filtration and migration method 60
3-3 Differentiation abilities of hADSCs 62
3-3.1 Osteogenic differentiation of isolated hADSCs (cell line, passage 5) by membrane migration method 63
3-3.2 Osteogenic differentiation of isolated hADSCs (Primary fat 83
3-3.3 Chondrogenic differentiation of isolated hADSCs (cell line, 92
3-3.4 Adipogenic differentiation of isolated hADSCs (cell line, 96
Chapter 4 Conclusions 98
Reference 101
Supplementary data 111
S-1 . Osteogenic differentiation of isolated hADSCs (cell line, passage 5) 113
S-2 . Osteogenic differentiation of isolated hADSCs (Primary) 121
S-3 . Adipogenic differentiation of isolated hADSCs (cell line, passage 5) 129
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2019-8-20
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