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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉逸軒(I-Hsuan Liu) | |
| dc.contributor.author | Dee-Shiuh Yang | en |
| dc.contributor.author | 楊迪旭 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:28:54Z | - |
| dc.date.available | 2023-08-23 | |
| dc.date.copyright | 2018-08-23 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-17 | |
| dc.identifier.citation | Armulik, A., A. Abramsson, and C. Betsholtz. 2005. Endothelial/pericyte interactions. Circ Res 97: 512-523.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78915 | - |
| dc.description.abstract | 間葉幹細胞 (mesenchymal stem cells，MSCs) 具有自我更新、免疫調節、多系分化及組織再生等功能，為細胞醫療產業極具潛力之角色。然而，能從體內分離出之MSCs數量有限，且細胞在體外培養過程中往往容易隨著繼代過程中逐漸失去原有之幹細胞特性 (stemness)，而大大降低與阻礙了其之運用。為了解決體外間葉幹細胞培養不易之問題，在我們先前的研究中發現，MSCs培養於去細胞 (decellularized) 之MS1內皮細胞胞外基質 (extracellular matrix，ECM) (MS1-ECM) 上，經過體外培養數代之後，相較於塑膠培養盤和自身之細胞外基質更能有效維持其幹細胞特性。本實驗中，為了能更有效率及方便利用MS1-ECM作為MSCs體外培養之生物材料，我們首先嘗試利用不同方法，包括：冷凍乾燥、酵素水解及超音波均質等方式處理MS1-ECM，再嘗試將所得之產物以細胞培養液添加物或培養盤塗層 (coating) 等兩種方式給予細胞。將初代MSCs培養於不同處理環境兩代後，藉由免疫調節試驗進行篩選，發現MS1-ECM利用超音波均質後再以培養盤塗層的方式應用，能有效提升MSCs的免疫調節指標基因的表現量。在電子顯微鏡下也可以觀察到，此種製備方式能有效重建原始MS1-ECM 之外觀型態。為了探討此一重建之MS1-ECM (reconstituted MS1-ECM，rECM) 能否為MSCs體外培養時帶來更大的益處，結合現行實務上常見之低氧 (5% O2) 培養條件，以進一步測試 rECM 維持初代MSCs特性之能力，包括：細胞數、細胞型態、表面抗原、自我更新能力及三系分化等。實驗結果發現，MSCs於常氧 (20% O2) 培養兩代後，rECM處理組相較於塑膠培養盤更能顯著維持正常型態、增進其細胞數量與脂肪、硬骨及軟骨的分化潛力，代表rECM的確能有效維持MSCs的特性。另一方面，MSCs於低氧下培養兩代後，較常氧更能顯著的增進細胞數量、自我更新及脂肪與軟骨分化能力，但硬骨分化能力卻顯著降低。此外，我們也發現MSCs若於低氧擴增後再移至常氧培養，似乎會降低細胞分化的潛力。為了進一步探究MS1-ECM中維持MSCs特性之重要分子組成，本研究也藉由蛋白質體學的方式，分析了MS1-ECM與MSC-ECM之蛋白質組成差異，期望能找出對MSCs特性維持的重要分子與其可能之機制與訊號路徑。 | zh_TW |
| dc.description.abstract | Mesenchymal stem cells (MSCs) have great potential for cell therapies. However, the scarcity and loss of stemness during amplification has been a challenge for their application. Our previous study indicated that decellularized extracellular matrix (ECM) derived from microvessel endothelial cell line Mile Sven 1 (MS1) can effectively preserve the MSC stemness including proliferation and differentiation capacities. In this study, we aimed to optimize the preparation and application of this biomaterial. We first tested various methods to harvest and homogenize MS1-ECM including lyophilization, enzymatic digestion and sonication, and also application methods including medium supplementation and plate coating. Screening by the response to interferon gamma stimulation, MSCs amplified on the plate coated by the sonicated MS1-ECM showed a dose-dependently significant increase. Furthermore, scanning electronic micrographs showed that MS1-ECM homogenized by sonication could successfully re-jellify on collagen-1-coated cultured plate and reconstitute the morphology of primary MS1-ECM (rECM). As hypoxia condition had gradually become the mainstream for expanding primary MSCs, we then evaluated the properties of MSCs amplified under normoxic (20% O2) or hypoxic (5% O2) culture conditions with or without rECM, and compared the cell numbers, morphology, immune-phenotypes as well as self-renewal and tri-linage differentiation capacities after two passages to assess the stemness preservation. The results indicated that rECM could better maintain MSCs stemness including proliferation and differentiation potentials compared to plastic plate control under normoxia. The major contents of MS1-ECM and MSC-ECM were further compared by proteomic analysis to dissect the potential factors that are critical for maintaining the stemness of BM-MSCs. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:28:54Z (GMT). No. of bitstreams: 1 ntu-107-R05626014-1.pdf: 14891943 bytes, checksum: 558a902dea8f836ae9d5ddfa44dcce8e (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iv List of figures vi List of tables viii Content ix Chapter 1 Introduction 1 1.1 Mesenchymal stem cells: history, properties and definition 1 1.2 Gold mine for cell therapy 2 1.2.1 MSCs-based cell therapy 2 1.2.2 Immuno-modulation of MSCs 3 1.3 Obstacles for MSCs application 6 1.3.1 Scarcity 6 1.3.2 Loss of stemness 6 1.4 Perivascular origin of MSCs 9 1.4.1 Hypothesis: pericytes as primitive MSCs 9 1.4.2 Interaction between endothelial cells and MSCs 9 1.5 A promise substrate for MSCs expansion: decellularization techniques of extracellular matrix 12 1.5.1 Decellularized-ECM from bone marrow cells 12 1.5.2 Decellularized-ECM from endothelial cells 13 1.5.3 Other sources of decellularized ECM used for MSCs expansion 15 1.6 ECM play an important role in modulating MSCs fates 16 1.6.1 Bio-physical environment of ECM 16 1.6.2 Bio-chemical components of ECM 17 1.7 Green house for MSCs: hypoxia culture condition 18 Chapter 2 Specific aims and experimental design 21 2.1 Aim I--- Finding out and standardize the proper application of MS1-ECM 21 2.2 Aim II--- Evaluation the stemness under normoxia and hypoxia condition with MS1-ECM coating 22 2.3 Aim III--- Proteomic analysis of MSC-ECM and MS1-ECM 23 Chapter 3 Materials and methods 26 3.1 Animals 26 3.2 Isolation and culture of bone marrow-derived mesenchymal stem cells 26 3.3 Culture of Mile Sven 1 (MS1) cell line 30 3.4 Cell passaging 30 3.5 Harvest of primitive decellularized MS1-ECM 30 3.6 Preparation for collagen I-coated 6 well plate 31 3.7 Preparation of decellularized MS1-ECM plates 31 3.8 Homogenization of fresh decellularized MS1-ECM 33 3.9 Protein quantification 33 3.10 Preparation of decellularized MS1-ECM coated plates 34 3.11 Lyophilization of fresh MS1-ECM 34 3.12 Homogenization of lyophilized MS1-ECM 34 3.13 Enzymatic digestion of lyophilized MS1-ECM 35 3.14 Coating MS1-ECM hydrogel on culture plate 35 3.15 Supplementing lyophilized and protease digested MS1-ECM 35 3.16 Immune-modulatory assay 36 3.17 Scanning electron microscope 36 3.18 Quantification of short/long axis ratio 37 3.19 Flow cytometric analysis 37 3.20 Colony-forming Unit (CFU) assay 39 3.21 Adipogenic differentiation 39 3.22 Osteogenic differentiation 40 3.23 Chondrogenic differentiation 41 3.24 Total RNA extraction 41 3.25 Reverse transcription PCR 42 3.26 Quantitative PCR 43 3.27 Proteomic analysis of ECM samples 45 3.28 Statistical analysis 45 Chapter 4 Results 47 4.1 Finding the new preparation method for MS1-ECM 47 4.1.1 Lyophilization and ball milled method 47 4.1.2 Lyophilization and enzymatic digestion method 49 4.1.3 Sonication and plate coating method 51 4.1.4 Assessment of the different preparation to determine the best preparation method 53 4.1.4.1 Sonicated MS1-ECM coating preparation recover the immune-modulation of MSCs 53 4.1.4.2 Sonicated MS1-ECM coating preparation had similar microenvironment of primary MS1-ECM 55 4.1.4.3 Sonicated MS1-ECM coating preparation had dose-effect in response to immune-modulation ability of MSCs 58 4.2 Comparison of sonicated MS1-ECM coating preparation and hypoxia culture in preserve MSC stemness 60 4.2.1 Plastic culture plate could not preserve morphology of MSCs under normoxia condition. 60 4.2.2 Sonicated MS1-ECM coating preparation promoted MSCs expansion under normoxia condition 66 4.2.3 Immune-phenotype of MSCs did not dramatically differ in each groups 68 4.2.4 Hypoxia condition significantly enhanced self-renewal ability of MSCs. 70 4.2.5 MSCs on sonicated MS1-ECM preserved the adipogenic differentiation potential 73 4.2.6 Sonicated MS1-ECM coating preparation preserved osteogenic differentiation potential whereas hypoxia condition decreased it 77 4.2.7 Sonicated MS1-ECM coating preparation preserve chondrogenic potential under normoxia and hypoxia condition 80 4.3 Dissecting and comparing the components of MS1-ECM and MSC-ECM 83 4.3.1 Proteomic composition and characterization of MSC-ECM and MS1-ECM 83 4.3.2 Characterization and quantification of ECM proteins presented in both MSC-ECM and MS1-ECM 86 4.3.3 Characterization and quantification of ECM proteins only presented in MSC-ECM 89 4.3.4 Characterization and quantification of ECM proteins only presented in MS1-ECM 92 Chapter 5 Discussion 94 5.1 Other sources of decellularized ECM used for MSCs expansion 94 5.2 Other preparation methods of ECM for MSCs to enhance their performance 94 5.3 MS1-ECM coating preparation partially recovered the beneficial effects of primary MS1-ECM 95 5.4 Different properties of MSCs reacted to external oxygenic tension change 97 5.5 Application of MS1-ECM coating preparation 99 5.6 Proteomic characterizations and differences between MSC-ECM and MS1-ECM 100 5.7 Quantities or qualities? How to use MS1-ECM as bio-materials for commercial MSCs culture? 101 5.8 Does MS1-ECM properly serve as the real vascular basement membrane niche for MSCs in vitro culture? 102 5.9 Why does sonicated MS1-ECM coating preparation have dose-dependent effect in enhancing the immune-modulatory activities of MSCs? 104 Chapter 6 Conclusion 105 口試問題回答 107 REFERENCE 114 | |
| dc.language.iso | en | |
| dc.subject | 低氧培養 | zh_TW |
| dc.subject | 內皮細胞 | zh_TW |
| dc.subject | 幹細胞 | zh_TW |
| dc.subject | 幹細胞特性 | zh_TW |
| dc.subject | 細胞外基質 | zh_TW |
| dc.subject | 生物材料 | zh_TW |
| dc.subject | 間葉幹細胞 | zh_TW |
| dc.subject | bio-materia | en |
| dc.subject | mesenchymal stem cells | en |
| dc.subject | pericytes | en |
| dc.subject | stem cell niche | en |
| dc.subject | decellularized extracellular matrix | en |
| dc.subject | hypoxia culture | en |
| dc.title | 運用MS1內皮細胞株所衍生之胞外基質作為擴增骨髓間葉幹細胞之生物材料 | zh_TW |
| dc.title | Optimizing the Preparation of MS1 Cells-derived Extracellular Matrix for the Amplification of Bone Marrow-derived Mesenchymal Stem Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃翠琴(Tsui-Chin Huang),皇甫維君(Wei-Chun HuangFu),林?輝(Feng-Huei Lin),林原佑(Yuan-Yu Lin) | |
| dc.subject.keyword | 間葉幹細胞,幹細胞特性,幹細胞,內皮細胞,細胞外基質,低氧培養,生物材料, | zh_TW |
| dc.subject.keyword | mesenchymal stem cells,pericytes,stem cell niche,decellularized extracellular matrix,hypoxia culture,bio-materia, | en |
| dc.relation.page | 126 | |
| dc.identifier.doi | 10.6342/NTU201803944 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-18 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-23 | - |
| 顯示於系所單位： | 動物科學技術學系 | |
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