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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳信志(Shinn-Chih Wu) | |
dc.contributor.author | Chun-Chun Cheng | en |
dc.contributor.author | 鄭淳淳 | zh_TW |
dc.date.accessioned | 2021-06-16T17:16:43Z | - |
dc.date.available | 2015-08-20 | |
dc.date.copyright | 2012-08-20 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63700 | - |
dc.description.abstract | 間葉幹細胞 (mesenchymal stem cell; MSC) 已知兼具自我更新 (self-renewal) 和複能性分化潛能 (multipotency) 等特性,因此在細胞治療與再生醫學,分別富具應用潛力。根據先前研究,骨髓中富含間葉幹細胞,而小鼠為接近人類之哺乳類動物且具有詳盡的基因背景資訊,因此多以此物種為廣泛研究之人類疾病模式動物。然而小鼠骨髓間葉幹細胞相較於其他物種而言,屬較難以分離與培養者,因而導致研究材料取得之不易,研究也因此易受侷限。本研究旨在由長骨之骨髓外組織來源分離具高增殖及分化能力之可替代骨髓間葉幹細胞的幹細胞。試驗首先自6至8週齡小鼠之骨骺端 (epiphysis) 成功分離並完成建立一新型之間葉幹細胞,命名為EMSC (epiphysis-derived MSC)。此等EMSC於培養過程中,不僅充分具備骨髓來源間葉幹細胞 (bone marrow-derived MSC; BMMSC) 固有之貼附性,且其群落形成 (clonogenic formation) 與細胞增生 (cell proliferation) 等能力,分別皆優於BMMSC者。此外,在誘導分化之特定培養條件下,此等EMSC充分具備能力分化成脂肪細胞、硬骨細胞與軟骨細胞,顯示其具備複能分化潛能。進一步針對細胞表面抗原分析結果顯示,此等EMSC不僅帶有包括CD29, CD44, CD73, CD105, CD166, Sca-1與SSEA-4等表面抗原;且不帶有CD11b, CD31, CD34, 與CD45等表面抗原。此外,在前述培養條件下,此等EMSC不表現主要組織相容性複合體I (MHC I) 與 MHC II,並有效抑制因異體免疫細胞刺激與刀豆素A刺激所導致之免疫細胞增生現象; 且能有效抑制IL-1, IL-6, 與TNF-alpha 等發炎細胞激素之產生,遂亦具有抗發炎之能力。而於細胞移行 (migration) 相關之化學激素 (chemokine) 受體表現分析試驗中顯示,EMSC主要為表現CXCR7,而BMMSC主要表現為CXCR4。又此等EMSC於低氧環境培養之下,可增強血管新生相關因子之表現能力,包括VEGF、Flk-1與PDGF。因受損組織為一低氧環境,藉由此等細胞之此特性,將可提升移植細胞治療時血管新生能力,以促進組織之修復過程。因此,進一步藉由活體內動物模式測試研究結果,證明EMSC在骨折試驗中,確能有效提升骨再生修復效果;於背部皮膚缺血 (skin flap) 之試驗結果,證明EMSC不僅可以顯著降低皮膚壞死區域,且有顯著提升皮膚血管新生之能力;並於後肢缺血試驗中且證明,EMSC可藉由幫助此處血管之再生,從而有效促進了血流的恢復。綜合上述試驗結果,顯示源自骨骺端所建立之新型間葉幹細胞– EMSC,細胞增生與分化能力均遠優於源自BMMSC者。此外,進一步體內活體試驗結果,亦證實其充分具備治療疾病之潛能。本研究結果顯示,此等源自骨骺端之間葉幹細胞殊具發展成為BMMSC替代來源之潛力,未來將有利於小鼠間葉幹細胞相關研究之推進。 | zh_TW |
dc.description.abstract | While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find stem cells of high proliferation and differentiation capacity as an alternative murine MSCs source from long bone except marrow tissue. In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-alpga in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. For chemokine receptors analysis, EMSCs expressed CXCR7 while BMMSCs expressed CXCR4. Furthermore, EMSCs significantly enhanced VEGF, Flk-1 and PDGF expression under hypoxia condition. The results showed that EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. These results indicate that EMSCs, a new type of MSCs, are a preferable alternative for murine MSCs due to their better proliferation and differentiation potentialities. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:16:43Z (GMT). No. of bitstreams: 1 ntu-101-D96642002-1.pdf: 11426247 bytes, checksum: c8b38eaa6aaf4b1ccca4759205517ed3 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | CONTENTS
致謝 I 中文摘要 II ABSTRACT IV CONTENTS V LIST OF FIGURES XI LIST OF TABLES XIII CHAPTER 1 LITERATURES REVIEW 1 1.1 Overview of Mensenchymal Stem Cells 1 1.1.1 History of MSCs Discovery 1 1.1.2 The Role of MSCs in Physiology 3 1.1.3 The Effects of MSCs on Immune Cells 6 1.1.4 Mechanisms of Immunosuppression by MSCs 11 1.1.5 Migratory Features of MSCs 12 1.1.6 Clinical Studies 13 1.2 The Stem Cell Niches in Bone 13 1.2.1 The Osteoblastic Niche on the Endosteal Surface 13 1.2.2 The Vascular Niche 15 1.2.3 The Role of Oxygen Availability in Stem Cell Function 15 1.2.4 Oxygen Levels Influence Stem Cell Phenotypes 16 1.3 Overview of Long Bone Development 17 1.3.1 Endochondral Ossification 17 CHAPTER 2 HYPOTHESIS 21 CHAPTER 3 DISCOVERY OF THE NOVEL EPIPHYSIS-DERIVED MSCS (EMSCS) 22 3.1 Introduction 22 3.2 Materials and Methods 22 3.2.1 Animals 22 3.2.2 Isolation and Culture of EMSCs 23 3.2.3 Isolation and Culture of Bone Marrow-Derived MSCs (BMMSCs) 24 3.2.4 EMSCs and BMMSCs Purification by the Transient Lower-Density Plastic Adherence (tLDA) Method 25 3.2.5 Flow Cytometric Analysis 25 3.2.6 Statistical Analysis 27 3.3 Results 27 3.3.1 Establishment of EMSCs and Compared to BMMSCs 27 3.3.2 Characterization of EMSCs 30 CHAPTER 4 EMSCS HAVE BETTER CELL PROLIFERATION AND DIFFERENTIATION POTENTIALS THAN BMMSCS 34 4.1 Introduction 34 4.2 Materials and Methods 34 4.2.1 Colony Formation Assay 34 4.2.2 Population Doubling Time (PDT) 35 4.2.3 Quantification of Telomere Length 35 4.2.4 Telomerase Activity 36 4.2.5 In Vitro Differentiation 37 4.2.6 Quantitative Real Time Reverse Transcription-Polymerase Chain Reaction (qPCR) 38 4.2.7 SA-β-Gal Staining 40 4.2.8 Statistical Analysis 40 4.3 Results 40 4.3.1 EMSCs Have Better Proliferation Potential than BMMSCs 40 4.3.2 EMSCs Show Higher Multipotent Potentials than BMMSCs 44 4.3.3 Senescence of EMSCs and BMMSCs during Culture 48 CHAPTER 5 EMSCS ARE IMMUNOSUPPRESSIVE AND ANTI-INFLAMMATORY 50 5.1 Introduction 50 5.2 Materials and Methods 50 5.2.1 Mitogen Proliferation Assays and Allogeneic Mixed Lymphocyte Reaction (MLR) 50 5.2.2 Transwell and Coculture Assay 51 5.2.3 Quantitative Real Time Reverse Transcription-Polymerase Chain Reaction (qPCR) 52 5.2.4 Statistical Analysis 53 5.3 Results 53 5.3.1 EMSCs Have Immunosuppressive Potential 53 5.3.2 EMSCs Have Anti-inflammatory Actions 53 CHAPTER 6 HYPOXIA EFFECTS ON EMSCS AND BMMSCS 56 6.1 Introduction 56 6.2 Materials and Methods 56 6.2.1 Quantitative Real Time Reversed Transcription-Polymerase Chain Reaction (qPCR) 56 6.2.2 Western Blot Analysis 59 6.2.3 Statistical Analysis 59 6.3 Results 60 6.3.1 Gene Expression Profiles of EMSCs and BMMSCs under Hypoxia 60 6.3.2 HIF-1a and HIF-2a expression of EMSCs and BMMSCs under Hypoxia 62 CHAPTER 7 THERAPEUTIC POTENTIALS OF EMSCS 64 7.1 Introduction 64 7.2 Materials and Methods 64 7.2.1 Bone Fracture Model 64 7.2.2 Skin Flap Model 65 7.2.3 Hindlimb Ischemia Model 65 7.2.4 Immunohistochemical Staining 66 7.2.5 Statistical Analysis 67 7.3 Results 67 7.3.1 EMSCs Enhance Fracture Repair 67 7.3.2 EMSCs Ameliorate Ischemia Skin Flap Necrosis 69 7.3.3 EMSCs Improve Blood Perfusion in Hindlimb Ischemia 71 CHAPTER 8 DISCUSSIONS AND CONCLUSIONS 73 REFERENCES 80 PUBLICATION LISTS 93 APPENDIX I 95 APPENDIX II 100 | |
dc.language.iso | en | |
dc.title | 骨骺來源間葉幹細胞之建立與特色分析及其治療應用潛能 | zh_TW |
dc.title | Establishment and Characterization of a Novel Mesenchymal Stem Cell Derived from Epiphysis and Their Potentials for Therapeutic Applications | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 鄭登貴(Teng-Kuei Cheng winstoncheng@ntu.edu.tw ) | |
dc.subject.keyword | 小鼠,骨髓,骨骺,間葉幹細胞,骨折,缺血, | zh_TW |
dc.subject.keyword | mouse,bone marrow,mesenchymal stem cells,immunosuppressive,bone fracture,skin flap,hindlimb ischemia, | en |
dc.relation.page | 129 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-18 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 生物科技研究所 | zh_TW |
顯示於系所單位: | 生物科技研究所 |
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