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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉逸軒(I-Hsuan Liu) | |
| dc.contributor.author | Yen-Hua Lee | en |
| dc.contributor.author | 李妍樺 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:39:12Z | - |
| dc.date.available | 2021-07-10T21:39:12Z | - |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-14 | |
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Sousa, S., Afonso, N., Bensimon-Brito, A., Fonseca, M., Simoes, M., Leon, J., Roehl, H., Cancela, M.L., and Jacinto, A. (2011). Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration. Development 138, 3897-3905. Stallcup, W.B. (2002). The NG2 proteoglycan: past insights and future prospects. J Neurocytol 31, 423-435. Stegmuller, J., Werner, H., Nave, K.A., and Trotter, J. (2003a). The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells - Implications for glial-neuronal signaling. Journal of Biological Chemistry 278, 3590-3598. Stegmuller, J., Werner, H., Nave, K.A., and Trotter, J. (2003b). The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial pr | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76876 | - |
| dc.description.abstract | 細胞命運決定為胚胎發育或組織再生過程中，決定細胞增生、遷移分化成最終形態之重要過程。了解細胞命運之決定機制可提升細胞療法於再生醫學之臨床應用。細胞命運決定之機制可受細胞內源及外源因子調控。其中硫酸軟骨素醣胺多醣、硫酸肝素蛋白多醣等蛋白多醣為調控細胞命運之重要外源因子。由於這些蛋白多醣和生長因子或型態生成誘導物間有高度親和力，可作為輔助受體或協助形成型態生成誘導物之濃度梯度，以影響細胞命運決定。本研究首先探討硫酸肝素分解酵素「硫酸肝素酶」對骨髓間葉幹細胞命運決定之影響。利用硫酸肝素酶抑制劑OGT2115抑制硫酸肝素酶之酵素活性，並檢測其對間葉幹細胞增生、聚落形成、分化及遷移之影響。結果顯示，當抑制間葉幹細胞內源硫酸肝素酶活性，細胞外硫酸肝素堆積增加，細胞增殖、聚落形成及軟骨分化能力降低，但可透過增強SDF-1/CXCR4訊息傳遞路徑增加細胞遷移行為。 上述研究證實細胞內源硫酸肝素酶活性對間葉幹細胞體外培養細胞命運決定扮演重要角色，但其在體內之真實調控機制仍待釐清。因此，若能產製標定間葉幹細胞之模式動物，有助於研究間葉幹細胞於體內之命運決定機制。先前研究顯示，間葉幹細胞位於血管周圍，且和血管周邊細胞具有類似特性。本研究嘗試以血管周邊細胞標誌基因「硫酸軟骨素蛋白多醣四」(chondroitin sulfate proteoglycan 4，Cspg4)於斑馬魚標定間葉幹細胞，以研究間葉幹細胞之活體功能。結果顯示，在Tg(Cspg4:Gal4; UAS:EGFP)基因轉殖斑馬魚胚及成魚尾鰭皆可觀察到帶有綠色螢光之細胞(Cspg4+)分佈於血管周邊。當基因轉殖斑馬魚尾鰭切除後，綠色螢光細胞(Cspg4+)數目增加，且多分佈於新生之尾鰭組織，當尾鰭再生至原有長度後，綠色螢光細胞(Cspg4+)逐漸消失，顯示這些細胞與組織再生有密切關聯。 最後，本研究進一步探討硫酸軟骨素蛋白多醣四於斑馬魚胚胎發育時期之功能。在胚胎發育時期調降硫酸軟骨素蛋白多醣四之表現量，會造成魚胚體軸過短。而此發育異常現象，可透過過量表現wnt11f2恢復正常。表示硫酸軟骨素蛋白多醣四可能透過調控Wnt/平面細胞極化通路影響體軸發育。此外，還發現當魚胚過量表現硫酸軟骨素蛋白多醣四會造成低比例的獨眼畸形，而硫酸軟骨素蛋白多醣四之穿膜結構域去除後也會造成此現象，表示硫酸軟骨素蛋白多醣四之分佈位置對於胚胎中線發育十分重要。 綜而言之，幹細胞或前驅細胞產生之硫酸肝素分解酵素「硫酸肝素酶」及細胞表面之硫酸軟骨素蛋白多醣四可調控細胞增生遷移、影響胚胎發育，為幹細胞命運決定機制中之重要調控因子。且本研究產製之Tg(Cspg4:Gal4; UAS:EGFP)基因轉殖斑馬魚可作為探討間葉幹細胞功能之有力工具，未來可利用此轉基因魚進一步研究硫酸肝素酶於活體中對幹細胞命運決定之影響。 | zh_TW |
| dc.description.abstract | Cell fate determination is the key step controlling cellular proliferation, differentiation and migration during embryogenesis and tissue regeneration. Understanding the mechanism of cell fate determination can improve the clinical application of cell therapy in regenerative medicine. Cell fate determination is regulated by both intrinsic and extrinsic mechanism. Extracellular matrix (ECM) is one of the important extrinsic factors regulating cell fate. One of the main components of ECM is proteoglycan, for example heparan sulfate proteoglycans and chondroitin sulfate proteoglycans. Proteoglycans have high affinity with growth factors and morphogens and consequently can act as a co-receptor or shape the morphogen gradient to regulate cell fate. In the first part of my study, the function of a heparan sulfate-degrading enzyme “heparanase” in bone marrow-derived mesenchymal stem cells (MSCs) fate determination was studied. I used a heparanase inhibitor “OGT2115” to inhibit the enzyme activity of cell-autonomous heparanase and tested the ability of cell proliferation, tri-lineage differentiation and migration of MSCs. The results showed that inhibiting cell autonomous heparanase increased the heparan sulfate deposition in the culture of MSCs, suppressed the cell proliferate, colony-forming and chondrogenic ability, and augmented the migratory behavior of MSCs by potentiating SDF-1/CXCR4 signaling axis. Although I found the heparanase as one of the key components regulating MSC fate in vitro, the endogenous niche of MSC is still not elucidated. Therefore, in the second part of my study, I tried to generate a transgenic zebrafish labeling MSCs to study the cell fate determination during embryogenesis and tissue regeneration in vivo. Previous studies suggested MSCs reside in the perivascular niche and have similar properties to perivascular cells. So I used a perivascular marker chondroitin sulfate proteoglycan 4 (Cspg4) as a marker and generated the Tg(Cspg4:Gal4; UAS:EGFP) transgenic zebrafish to study the MSCs in vivo. The results demonstrated that Cspg4+ cells wrap around the blood vessels in the zebrafish embryos and adult caudal fin. During caudal fin regeneration, theses Cspg4+ cells increased and migrated to the injured site and largely disappeared after fin regeneration indicating that Cspg4+ cells might contribute to tissue regeneration. In the third part of my study, I further evaluated the cellular function of the MSC marker Cspg4 in zebrafish by knocking down cspg4 in zebrafish embryos. The result demonstrated that knocking-down cspg4 resulted in a shorter anterior-posterior axis compared to control embryo, which could be rescued by co-injecting wnt11f2 mRNA suggesting that Cspg4 regulates body axis organization through modulating the Wnt/planar cell polarity signaling pathway. In addition, overexpressing cspg4 caused cyclopia. The Cspg4 transmembrane domain mutant embryo phenocopied the global overexpression of cspg4 mRNA and led to 8% cyclopia. These results demonstrated that the quantitative and spatial accuracy of Cspg4 expression is critical for body axis and midline development during gastrulation. In summary, the Cspg4 on the cell surface and the heparan sulfate degrading enzyme heparanase contribute to cell fate determination of stem cells or progenitor cells. The Tg(Cspg4:Gal4; UAS:EGFP) transgenic zebrafish could be a useful model to study the in vivo roles of heparanase and Cspg4 in MSC fate determination. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:39:12Z (GMT). No. of bitstreams: 1 U0001-1208202014233300.pdf: 3824821 bytes, checksum: ec4b634804f1ebf92f08e880a9a887e1 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | CHAPTER 1. HEPARAN SULFATE PROTEOGLYCANS AND CHONDROITIN SULFATE PROTEOGLYCANS IN MESENCHYMAL STEM CELLS 1 1.1. STEM CELLS 1 1.1.1. Mesenchymal stem cells 2 1.1.2. Clinical application of mesenchymal stem cells 3 1.1.3. The markers of mesenchymal stem cells 4 1.1.4. The perivascular niche of mesenchymal stem cells 7 1.2. PROTEOGLYCANS IN STEM CELL FATE DETERMINATION 9 1.2.1. Heparan sulfate proteoglycans 10 1.2.2. Heparanase 12 1.2.3. Chondroitin sulfate proteoglycans 13 1.3. SPECIFIC AIMS OF THE THESIS 14 CHAPTER 2. THE PHYSIOLOGICAL ROLE OF HEPANRANASE IN MOUSE BONE MARROW MESENCHYMAL STEM CELLS 15 2.1. INTRODUCTION 15 2.2. HYPOTHESIS 16 2.3. MATERIAL AND METHODS 16 2.3.1. Experimental animal 16 2.3.2. Isolation of mouse bone marrow MSCs 16 2.3.3. RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) …………………………………………………………………………………………18 2.3.4. Immunocytochemistry 18 2.3.5. Heparanase activity 19 2.3.6. Preparation of mouse recombinant heparanase 20 2.3.7. In vitro differentiation 21 2.3.8. Quantitative polymerase chain reaction (qPCR) 24 2.3.9. MTT (3-(4, 5-dimethylithiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay …………………………………………………………………………………………………………………25 2.3.10. Colony forming assay 25 2.3.11. Transwell cell migration assay 26 2.3.12. DNA Topoisomerase assay 26 2.3.13. Statistic analysis 27 2.4. RESULTS 28 2.4.1. BM-MSCs express enzymatically active Hpse1 28 2.4.2. The effect of heparanase inhibitor on tri-lineage differentiation 31 2.4.3. Heparanase modulates proliferation, clonogenic ability of MSCs 35 2.4.4. Heparanase regulates cell migration ability of MSCs 37 2.4.5. The effect of heparanase activity in topoisomerase 39 2.5. DISCUSSION 41 CHAPTER 3. USING CSPG4 AS A MARKER TO LABEL MESENCHYMAL STEM CELLS IN ZEBRAFISH 44 3.1. INTRODUCTION 44 3.2. HYPOTHESIS 45 3.3. MATERIAL AND METHODS 45 3.3.1. Zebrafish maintenance and egg collection 45 3.3.2. Generation of Tg(cspg4:Gal4; UAS:EGFP) zebrafish 46 3.3.3. Angiography of zebrafish embryo 46 3.3.4. Angiography of adult zebrafish 47 3.3.5. Zebrafish caudal fin amputation 47 3.4. RESULTS 48 3.4.1. Cspg4+ cells reside in the perivascular niche 48 3.4.2. The dynamics of Cspg4+ cells during zebrafish tail fin regeneration 52 3.5. DISCUSSION 56 CHAPTER 4. THE ROLES OF CSPG4 DURING ZEBRAFISH EMBRYO DEVELOPMENT 59 4.1. INTRODUCTION 59 4.2. HYPOTHESIS 60 4.3. MATERIAL AND METHODS 60 4.3.1. Experimental animal 60 4.3.2. Temporal and spatial expression pattern of cspg4 60 4.3.3. Molecular cloning 63 4.3.4. Morpholino and mRNA microinjection 64 4.3.5. Alcian blue staining 65 4.3.6. Generation of Cspg4 mutant line by using CRISPR/Cas9 system 65 4.3.7. Genotyping of Cspg4 mutant line 67 4.3.8. Western blot 67 4.3.9. Immunoprecipitation 68 4.3.10. Statistic analysis 69 4.4. RESULTS 70 4.4.1. Cspg4 is expressed during zebrafish embryogenesis 70 4.4.2. Zebrafish body axis elongation and pharyngeal cartilage patterning required Cspg4 76 4.4.3. The location of Cspg4 is important for midline development 86 4.4.4. Cspg4 regulates body axis organization by Wnt11f2 pathway 89 4.5. DISCUSSION 97 CHAPTER 5. CONCLUSIONS AND PERSPECTIVE 101 REFERENCES 102 APPENDIX 119 | |
| dc.language.iso | en | |
| dc.subject | 胚胎發育 | zh_TW |
| dc.subject | 間葉幹細胞 | zh_TW |
| dc.subject | 硫酸肝素酶 | zh_TW |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | 硫酸軟骨素蛋白多醣四 | zh_TW |
| dc.subject | 再生 | zh_TW |
| dc.subject | 原腸胚形成 | zh_TW |
| dc.subject | mesenchymal stem cells | en |
| dc.subject | gastrulation | en |
| dc.subject | embryonic development | en |
| dc.subject | zebrafish | en |
| dc.subject | fin regeneration | en |
| dc.subject | chondroitin sulfate proteoglycan 4 | en |
| dc.subject | heparanase | en |
| dc.title | 蛋白多醣於幹細胞命運決定之角色 | zh_TW |
| dc.title | The Roles of Proteoglycans in Stem Cell Fate Determination | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 李士傑(Shyh-Jye Lee),林劭品(Shau-Ping Lin),陳全木(Chuan-Mu Chen),陳振輝(Chen-Hui Chen),皇甫維君(Wei-Chun HuangFu) | |
| dc.subject.keyword | 間葉幹細胞,硫酸肝素酶,斑馬魚,硫酸軟骨素蛋白多醣四,再生,原腸胚形成,胚胎發育, | zh_TW |
| dc.subject.keyword | mesenchymal stem cells,heparanase,chondroitin sulfate proteoglycan 4,fin regeneration,zebrafish,embryonic development,gastrulation, | en |
| dc.relation.page | 127 | |
| dc.identifier.doi | 10.6342/NTU202003084 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-17 | |
| 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 |
| 顯示於系所單位： | 動物科學技術學系 | |
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