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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻(Tai-Horng Young) | |
dc.contributor.author | Chien-Hsun Huang | en |
dc.contributor.author | 黃建勛 | zh_TW |
dc.date.accessioned | 2021-06-15T04:45:54Z | - |
dc.date.available | 2014-08-16 | |
dc.date.copyright | 2010-08-16 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-05 | |
dc.identifier.citation | Reference List
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45773 | - |
dc.description.abstract | 成體幹細胞是一群未分化完全的細胞,同時具有分裂及自我增生、分化成為具多種特定功能體細胞的特性。人類間葉幹細胞已知是目前用途最廣的多分化性細胞,大部份具有間葉細胞特性的細胞群,包含骨細胞、肌細胞、軟骨細胞、脂肪細胞等等,都可以由間葉幹細胞的分化而來。此外,間葉幹細胞也已經被證實富含於各組織中,包括肌肉、脂肪、臍帶、肝、肺、關節腔滑膜及牙髓組織等,都已經有報告指出可以分離出間葉幹細胞。
牙髓幹細胞具有很好的形成細胞群落、分裂及自我增生、及多重分化的能力,因此在硬組織再生工程中,是非常理想的細胞來源。在本篇研究的第一部份,由第三大臼齒的牙髓組織所分離培養的牙髓細胞,在經過數次的繼代培養後,經由流式細胞儀,分選出帶有及不帶有Stro-1及CD146表面抗原的細胞群。本研究中,帶有Stro-1及CD146表面抗原的細胞群定義為牙髓幹細胞 (DPSCs);而不帶有的則定義為牙髓細胞 (DPCs)。值得注意的是,牙髓幹細胞表現出較牙髓細胞更好的形成群落特性及成骨分化的能力。而這些特性的差異,則與牙髓幹細胞內N-乙醯葡萄醣胺轉移酶-5 (N-acetylglucosaminyltransferase V, GnT-V)的表現較高有關。 進一步探討後可以發現,在系統存在不同的小分子量醣胺類衍生物時,例如葡萄醣胺(glucosamine)、N-乙醯葡萄醣胺(N-acetyl-d-glucosamine)、甘露醣胺(d-mannosamine)、與N-乙醯甘露醣胺(N-acetyl-d-mannosamine)等,牙髓幹細胞中的N-乙醯葡萄醣胺轉移酶-5可以催化N-端連結醣鏈(N-glycan)形成第一型轉化生長因子接受器 (transforming growth factor-β receptor type I)的產生,且增加的第一型轉化生長因子接受器可以進一步的活化其下游Smads的信號。且實驗結果發現,在0.005 mg/ml的濃度中,細胞的早期成骨分化會有明顯的提升。藉由調控N-乙醯葡萄醣胺轉移酶-5催化N-端連結醣鍊(N-glycan)形成第一型轉化生長因子接受器與其下游的Smads信號,被葡萄醣胺/N-乙醯葡萄醣胺、甘露醣胺/N-乙醯甘露醣胺刺激的牙髓幹細胞會進一步的提高成骨特異性轉錄因子(Runx2/Cbfa1)的磷酸化表現,及後期與成骨分化相關的基因表現,如: 鹼性磷酸酵素(ALP)及骨鈣蛋白(osteocalcin)等等。再者,小分子醣胺類衍生物所引起的牙髓幹細胞早期成骨分化,在幫助誘導成骨分化的補充劑存在下,能夠刺激細胞產生更多的礦物基質沉積。 總結本篇的研究的結果,牙髓幹細胞具有較佳的形成細胞群落與分裂及自我增生的能力,而此現象與N-乙醯葡萄醣胺轉移酶-5 (N-acetylglucosaminyltransferase V, GnT-V) 的表現較高有關。在外加的低分子量葡萄醣胺/N-乙醯葡萄醣胺、甘露醣胺/N-乙醯甘露醣胺刺激時,O-端連結的蛋白質醣基化表現並不明顯。相對地,N-乙醯葡萄醣胺轉移酶可進一步增加N-端連結醣鏈形成的第一型轉化生長因子接受器的表現,且經由Smads的信號傳遞下,達到調控牙髓幹細胞成骨分化的效果。 | zh_TW |
dc.description.abstract | Adult stem cells are undifferentiated cells, which have the abilities to divide or self-renew indefinitely and generate most cell lineages. Human mesenchymal stem cells (MSCs) are known to be most useful multipotent cells capable of differentiating into various mesenchymal phenotypes, including osteogenic, myogenic, chrondogenic, and adipogenic lineages. Furthermore, MSCs have also been verified that they abound in various tissues and are able to be isolated from skeletal muscle, adipose tissue, umbilical cord, liver, lung, synovium as well as dental pulp.
Dental pulp stem cells (DPSCs) are ideal cell source for hard tissue regeneration due to their well clonogenic, self-renewing, and multi-potential capability. In the part I of this study, primary cell cultures were obtained from human dental pulp tissue of developing third molars, and flow cytometry was used to sort the subpopulation of DPSCs with STRO-1 and CD146 double-positive expression (denoted “DPSCs”). It was noted that DPSCs exhibited superior colonogenic potential and osteogenic differentiation capability to the dental pulp cell subpopulation with STRO-1 and CD146 double-negative expression (denoted DPCs). Besides, our recent study first revealed that the higher level of N-acetylglucosaminyltransferase V (GnT-V) resulted in the superior colonogenic and self-renewal potential of DPSCs. Furthermore, the GnT-V catalyzed the production of N-glycan-branching transforming growth factor-β receptor (TGFβr) type I, which subsequently activated Smads signals in DPSCs with various hexosamine derivatives, e.g. glucosamine (GlcN), N-acetyl-d-glucosamine (GlcNAc), d-mannosamine (ManN) and acetyl-d-mannosamine (ManNAc). A low concentration (0.005 mg/ml) of exogenous hexosamine derivatives was effective in promoting the early osteogenic differentiation of DPSCs. By modulating the GnT-V catalyzed N-glycan forming TGF-βr type I and subsequently Smads signal pathways, GlcN/GlcNAc and ManN/ManNAc treated DPSCs phosphorylated the Runt-related transcription factor 2/Core-binding factor alpha1 (Runx2/Cbfa1) and upregulated mRNA levels of Runx2/Cbfa1, alkaline phosphatase (ALP) and osteocalcin (OCN). In the presence of osteogenic supplements, hexosamine-treated DPSCs produced more mineralized-matrix deposition than did the untreated groups. In summary, this study demonstrated that DPSCs exhibited high level of Gnt-V, which resulted in the superior colonogenic and self-renewal potential of DPSCs. The exogenous hexosamine with low molecular weight, such as GlcN/GlcNAc and ManN/ManNAc, facilitated GnT-V catalyzed N-glycan branching glycoprotein on surface, e.g. TGFβr type I, rather than O-linked glycosylation; and increase level of TGFβr type I further regulated the osteogenic differentiation of human DPSCs through Smads signal pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:45:54Z (GMT). No. of bitstreams: 1 ntu-99-F92548034-1.pdf: 4436107 bytes, checksum: 943ccb5f15f2ef41ed8c1ea65d349d08 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | Contents..................................................I
List of tables............................................V List of Figures...........................................VI 摘要……………………………………………………………………XVII Abstract………………………………………………………………XIX Abbreviations…………………………………………………………XXI Chapter 1 Background ......................................1 1.1. Stem cells.........................................1 1.2. Adult stem cells...................................1 1.2.1 Mesenchymal stem cells (MSCs)......................2 1.2.2 Dental pulp stem cells (DPSCs).....................3 1.3. Regulating factors of MSCs’ differentiation......4 1.3.1 Effects of insoluble factors.......................4 1.3.2 Effects of soluble factors.........................6 1.4. Glucosamine and protein glycosylation .............8 Chapter 2 Introduction and Motivations....................10 Chapter 3 Materials and Methods...........................16 3.1 Reagents and chemicals............................16 3.2 Culture of dental pulp stem cells.................17 3.3 Sorting of dental pulp stem cells by flow cytometry.................................................17 3.4 Assay of colony-forming efficiency and the cell proliferation rate........................................18 3.5 Preparation of GlcN/oligo-GlcN, hexosamine, osteogenic supplements, and SB431542......................18 3.6 Cell viability....................................19 3.7 RNA isolation and quantitative real-time PCR......19 3.8 ALP and mineralized matrix deposition.............20 3.9 Western blotting and immunoprecipitation..........21 3.10 Small Interfering RNA transfection................22 3.11 Statistical analyses..............................23 Chapter 4 Results.........................................24 4.1 Part I: Characteristics of Dental Pulp Stem Cells.24 4.1.1 Expressions of retained Stro-1 and CD146 on DPSCs.24 4.1.2 Expressions of surface antigen on DPSCs and DPCs..24 4.1.3 Colony colony-forming efficiencies, and proliferation rates of DPSCs and DPCs.....................25 4.1.4 Expression of glycosyltransferases in DPSCs and DPCs......................................................25 4.1.5 Colony colony-forming efficiencies, and proliferation rates of Mgat5 knockdown DPSCs..............25 4.2 Part II: Effects of Glucosamine and Hexosamine derivatives in Regulating Osteogenic Differentiation of Dental Pulp Stem Cells....................................27 4.2.1 Effects of GlcN/oligo-GlcN on the cellular viability and ALP activity of DPSCs.......................27 4.2.2 GlcN/oligo-GlcN upregulated the mRNA expression of osteogenic genes..........................................27 4.2.3 GlcN/oligo-GlcN increased the ALP activity and mineralized-matrix deposition.............................28 4.2.4 GlcN/GlcNAc and ManN/ManNAc up-regulated the mRNA expressions of osteogenic genes in DPSCs..................29 4.2.5 GlcN/GlcNAc and ManN/ManNAc up-regulated the the ALP activity and mineralized matrix deposition............29 4.3 Part III: Mechanism of Glucosamine and Hexosamine derivatives in Regulating Osteogenic Differentiation of Dental Pulp Stem Cells....................................31 4.3.1 GlcN upregulated the TGF-β receptor type I and Smad2 phosphorylation.....................................31 4.3.2 GlcN/GlcNAc and ManN/ManNAc elevated N-glycan branching TGF-β receptor type I and Smads signaling in DPSCs ....................................................31 4.3.3 Phosphorylated Runx2/Cbfa1by activated Smads protein...................................................32 4.3.4 SB431542 inhibited the TGF-β receptor type I and Smad2 phosphorylation.....................................32 4.3.5 GlcN enhanced the osteogenic differentiation of DPSCs through TGF-βr type I and Smad2 signaling..........33 4.3.6 The role of Gnt-V regulated N-glycan branching TGF-βr type I in osteogenic differentiation of DPSCs.....33 Chapter 5 Discussion......................................35 Chapter 6 Conclusions and Perspectives....................45 Chapter 7 References......................................46 Appendix 1 Effects of Cyclic Mechanical Stretching on the Regulation of Tendon/Ligament-Related and Osteoblast-Specific mRNA expression in Human Mesenchymal Stem Cells..87 Abstract..................................................87 1.1 Introduction.....................................88 1.2 Material and methods.............................90 1.2.1 Isolation and Growth of MSCs.....................90 1.2.2 Flow Cytometry...................................90 1.2.3 Application of Mechanical Stretching.............91 1.2.4 RNA isolation....................................91 1.2.5 Quantitative Real-Time PCR.......................92 1.3 Results..........................................93 1.3.1 Expressions of hMSCs surface antigens............93 1.3.2 Morphology and Orientation of hMSCs..............93 1.3.3 mRNA Expressions of MSCP and MMP-3...............94 1.3.4 mRNA Expression of Tendon/Ligament-Related Markers...................................................94 1.3.5 mRNA Expression of Osteoblast-Specific Markers...95 1.4 Discussion.......................................95 1.5 Conclusions......................................99 1.6 References......................................100 Appendix 2 Interactive Effects of Mechanical Stretching and Extracellular Matrix Proteins on Initiating Osteogenic Differentiation of Human Mesenchymal Stem Cells..........109 Abstract.................................................109 2.1 Introduction....................................110 2.2 Materials and methods...........................112 2.2.1 Cell culture.....................................113 2.2.2 Application of mechanical stretching to cultured cells....................................................113 2.2.3 Metabolic assay..................................114 2.2.4 Immunofluorescence staining......................114 2.2.5 RNA isolation and quantitative real-time polymerase chain reaction...........................................115 2.2.6 Immunoprecipitation and Western blot analysis....116 2.2.7 Measurement of alkaline phosphatase (ALP) activity.................................................117 2.2.8 Alizarin red S staining and quantitative assessment...............................................118 2.3 Results..........................................118 2.3.1 Characteristics of hMSCs........................118 2.3.2 Effect of mechanical stretching on MTT activity.119 2.3.3 Phosphorylation of FAK and ERK 1/2..............119 2.3.4 Expression and phosphorylation of Cbfa1.........120 2.3.5 ALP activity and ARS staining...................121 2.4 Discussion.......................................122 2.5 Conclusions......................................127 2.6 References.......................................128 List of publication......................................138 List of conference.......................................139 Introduction to Author...................................140 | |
dc.language.iso | en | |
dc.title | 己醣胺類衍生物對牙髓幹細胞成骨分化影響之研究 | zh_TW |
dc.title | Study of hexosamine derivatives on osteogenic differentiation of dental pulp stem cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 陳羿貞(Yi-Jane Chen) | |
dc.contributor.oralexamcommittee | 王盈錦(Yng-Jiin Wang),宋信文(Hsing-Wen Sung),方旭偉(Hsu-Wei Fang),鄭廖平(Liao-Ping Cheng),林俊彬(Chun-Pin Lin) | |
dc.subject.keyword | 牙髓幹細胞,葡萄糖胺,己醣胺,成骨分化,N-乙醯胺基葡萄糖轉移酶,轉化生長因子-β接受器,Smads信號蛋白, | zh_TW |
dc.subject.keyword | Dental pulp stem cells,Glucosamine, Hexosamine,Osteogenic differentiation,N-acetyl-glucosaminyltransferases,TGF-β receptor,Smads, | en |
dc.relation.page | 140 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-06 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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