博碩士論文 103324033 詳細資訊




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姓名 陳彥名(Yen-Ming 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 水凝膠嫁接不同序列寡肽用以無異種培養人類多功能幹細胞
(Hydrogels Grafted with Different Oligo-peptide Sequence for Xeno-free Culture of Human Pluripotent Stem Cells)
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摘要(中) 人類多功能幹細胞具有很高的治療疾病潛力,而利用合成的生醫材料培養幹細胞相較於飼養層的培養,提供了較低的成本、較高的再現性且無異種汙染問題。因此,本研究將人類胚胎幹細胞(WA09)及人類誘導多功能幹細胞(HPS0077)培養在具不同奈米片段的生醫材料上。本研究中,我們先在培養盤上覆蓋一層乙烯醇-衣康酸共聚物 (PVA-IA) 的高分子膜並將表面硬度調整為25.3 kPa後,在表面嫁接上源自細胞外基質的貼附細胞寡肽。這些寡肽取自不同的人類蛋白,有玻璃粘連蛋白 (vitronectin, KGGPQVTRGDVFTMP), 骨唾液酸蛋白 (bone sialoprotein, KGGNGEPRGDTYRAY), 肝素鍵結端多肽 (heparin binding domain peptide, GKKQRFRHRNRKG)。除了基本單鏈結構的寡肽外 (O-VN1),本研究也設計了長鏈 (O-VN2G, GGGGKGGPQVTRGDVFTMP)及雙鏈結構 (O-VN2C, GCGGKGGPQVTRGDVFTMP)。因為O-VN2C具有半胱氨酸 (cysteine) 所以在嫁接過程中會自動形成雙硫鍵,所以這個雙鏈序列會有較高的表面寡肽密度。此外,本研究也以二次活化及嫁接的方式設計了樹枝鏈結構。藉著活化第一次嫁接的寡肽序列,我們能將細胞貼覆寡肽嫁接於第一個序列上來形成樹枝鏈結構。藉由培養人類多功能幹細胞於不同改質後的表面來探討對細胞的不同影響。
摘要(英) Human pluripotent stem cells (hPSCs) have significant potential in therapeutic applications for many diseases. The feeder-free cultures using synthetic biomaterials as stem cell culture materials offer more reproducible culture conditions and lower the cost of production without introducing xenogeneic contaminants. Therefore, we investigated hESCs (H9) and human induced pluripotent stem cells (hiPSCs, HPS0077) culture on biomaterials grafted with different sequence of nanosegments.
We prepared dishes coated with polyvinylalcohol-co-itaconic acid (PVA-IA) hydrogels having optimal elasticity of 25.3 kPa storage modulus, and grafted with extracellular matrix (ECM)-derived cell-adhesion peptides. hPSCs were cultured in a chemically defined medium on PVA-IA hydrogels grafted with oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP), bone sialoprotein (KGGNGEPRGDTYRAY), heparin binding domain peptide (GKKQRFRHRNRKG), and also two other sequences that are modified from basic oligo-vitronectin, of which we call VN2C (GCGGKGGPQVTRGDVFTMP) and VN2G (GGGGKGGPQVTRGDVFTMP). VN2C contains cysteine (C) amino acid containing sulfur element, which allows that two VN2C peptides form a dimer structure by S-S bonding spontaneously in atmosphere, so this sequence of oligopeptide (VN2C) is expected to have a higher surface density of oligopeptides compared to other oligopeptide. Besides, we also design a branch type oligopeptide structure by using dual activation and grafting methods. Following the reaction of oligopeptide on PVA-IA hydrogels after the first time grafting, another oligopeptide was subsequently grafted to create a branch-like structure. hPSC were expanded on each nanobrush (oligopeptide) grafted hydrogels with different nanobrush design and discussed the optimal design of nanosegments.
關鍵字(中) ★ 幹細胞
★ 寡肽
★ 人類多功能幹細胞
★ 人類胚胎幹細胞
★ 人類誘導多功能幹細胞
★ 無異種培養
關鍵字(英) ★ stem cell
★ oligopeptide
★ human pluripotent stem cell
★ human embryonic stem cell
★ human induced pluripotent stem cell
★ xeno-free culture
論文目次 Outline
Introduction 1
Ⅰ-Ⅰ Stem Cells 1
I-I-I Stem Cells and Stem Cell Therapy 1
I-I-II Human Embryonic Stem Cells (hESCs) 2
I-I-III Human Induced Pluripotent Stem Cells (hiPSCs) 3
I-I-IV Human Multipotent Stem Cells 4
Ⅰ-Ⅱ Human Pluripotent Stem Cells Culture 5
I-II-I Materials for hPSCs Culture 5
I-II-I-I Extracellular Matrices (ECMs) Coated Surfaces 6
I-II-I-II Polysaccharide-immobilized Surfaces for hPSCs Culture 8
I-II-I-III Polymeric Materials for hPSCs Culture 9
I-II-I-III-I Synthetic Polymers 9
I-II-I-III-II Oligopeptide Immobilized Surfaces 11
I-II-I-III-III 3D culture of hPSCs 12
I-II-I-III-IV Thermoresponsive Materials for hPSCs Culture 13
Ⅰ-Ⅱ-Ⅱ Influence of Different Environment on hPSCs 14
Ⅰ-Ⅲ Characterization of Human Pluripotent Stem Cells 16
I-III-I Colony Formation 17
I-III-II Alkali Phosphatase Activity 18
I-III-III Pluripotent Gene Expression 18
I-III-IV Pluripotent Protein Expression 18
I-III-V Differentiation Ability 19
I-III-VI Immunofluorescence 20
Ⅱ. Material and Methods 22
Ⅱ-Ⅰ Material 22
II-I-I Cell Line 22
II-I-II Commercial Coated Dishes 22
II-I-III Medium and Others 22
II-I-IV Synthetic Material – PVA-IA Dishes 22
II-I-V Immunostaining 23
Ⅱ-Ⅱ Cell Culture 23
II-II-I Preparation of PVA-IA Cell Culture Surfaces 23
II-II-II XPS Analysis of Dish Surfaces 26
II-II-III Methods of Culturing and Passaging hPSCs 26
II-II-IV Cryopreservation of Human Pluripotent Stem Cells 27
II-II-V Thaw of Human Pluripotent Stem Cells 28
Ⅱ-III Characterization of Human Pluripotent Stem Cells 28
II-III-I Immunostaining 28
II-III-II Expansion Fold and Doubling Time of hPSCs 30
II-III-III Differentiation Ratio of hPSCs 30
II-III-IV Differentiation Ratio of hPSCs 31
II-III-V Embryoid Body Formation 31
II-III-VI Teratoma Formation 32
Ⅲ. Result and Discussion 33
Ⅲ-I Characterization of Oligopeptide-Grafted Surfaces 33
Ⅲ-II Cultivation of hPSCs on Oligopeptide-Grafted Surfaces 40
III-II-I Short Term Culture of hESCs: Condition Screening 40
III-II-II Long Term Culture of hPSCs 49
III-II-III Characterization of hPSCs after Long Term Culture 55
III-II-III-I Immunostaining of Pluripotent Genes 55
III-II-III-II Differentiation Ability In Vitro – Embryoid Body Formation 59
Ⅳ. Conclusions 64
Supplement Data 66
Supplement tables 66
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2016-7-21
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