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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68121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 | Ching-Wen Tsai | en |
| dc.contributor.author | 蔡靜雯 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:13:00Z | - |
| dc.date.available | 2023-01-04 | |
| dc.date.copyright | 2018-01-04 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-12-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68121 | - |
| dc.description.abstract | 一個適宜的體外腫瘤模型可促進抗癌藥物的開發與相關市場的發展。近年來，癌細胞與間葉幹細胞或纖維母細胞的三維共培養模型已逐漸被提出，其效果也被證實較二維或癌細胞單獨培養模型更貼近體內的腫瘤情形。生醫高分子幾丁聚醣能有效地調節間葉幹細胞與癌細胞的幹細胞特性，且能使細胞在培養時懸浮聚集成球。幾丁聚醣對於癌細胞、間葉幹細胞與纖維母細胞的幹細胞特性之調控，在此論文中透過CD44的蛋白表現被初步地檢驗。立基於以上數個幾丁聚醣特點，我們提出一個新穎的體外共培養且具核殼結構的多細胞球腫瘤模型。此腫瘤模型特點在於間葉幹細胞或纖維母細胞能聚集於多細胞球體之球核位置，癌細胞則包覆於外形成多細胞球體的球殼。球核內之間葉幹細胞或纖維母細胞能有效地支持球殼的癌細胞進而提升其細胞粒線體活性與對抗抗癌藥物的耐受性。再者，癌細胞也能保護間葉幹細胞或纖維母細胞，使其免於抗癌藥物的攻擊。
由實驗結果發現，幾丁聚醣的存在並不會影響纖維母細胞的CD44蛋白表現，只會調控具有幹細胞的‟自我更新”特性。因此，我們推測幾丁聚醣也能調節纖維母細胞的增殖能力而達到‟自我更新”的目的。當老化之人類包皮纖維母細胞培養在幾丁聚醣上時，細胞能懸浮聚集成球且同時延緩其老化，提升增殖能力與遷移能力。當纖維母細胞懸浮培養於聚乙烯醇或聚甲基丙烯酸羥乙基酯基材上時，並無延緩老化之效果。進一步分析細胞的老化相關蛋白表現，幾丁聚醣能有效地向下調節細胞內與培養環境中的轉化生長因子-β之表現。幾丁聚醣調控轉化生長因子-β的訊號傳遞，進而達到延緩細胞老化和提高其增殖能力的效果。最後，此模型也應用於人類膝關節腔內之前十字韌帶細胞與滑膜纖維母細胞。數據顯示，幾丁聚醣除有效地延緩細胞之老化外，也能提升其功能相關蛋白和遷移能力。 | zh_TW |
| dc.description.abstract | An ideal in vitro drug screening model is important for the drug development. In addition to monoculture systems, coculture systems have been used to mimic the in vivo tumor tissues because cell-cell and cell-extracellular matrix interactions can be studied. In this study, suspension core/shell coculture multicellular spheroids on chitosan are developed. Based on the characteristic of chitosan inhibiting cell adhesion, SW620 (colon cancer cell line), 3A6 (mesenchymal stem-like cell line) and Hs68 (foreskin fibroblast line) cells can aggregate to form 3D coculture spheroids with intimate cell contacts. CD44 is extensively expressed within adult bone marrow and has been considered as an important marker for cancer stem cells in some types of tumors. Therefore, it is used to understand the variations of stem cells and cancer cells when cells culturing on chitosan firstly. When cells are cocultured on chitosan, 3A6 and Hs68 cells always locate in the core of spheroids and are completely enveloped by SW620 cells following the differential adhesion hypothesis. Moreover, the core cells can stimulate the shell SW620 cells to enhance activity and resistance against the cytotoxicity effect of chemotherapy drugs. Therefore, based on the specificity of the core/shell coculture multicellular spheroids, a novel in vitro tumor model is proposed.
Fibroblasts have been extensively used as a model to study cellular senescence. The second purpose of this study is to investigate whether the human foreskin fibroblast aging process can be regulated by using the chitosan. Senescent cells are collected and seeded on chitosan to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in multicellular spheroids are downregulated significantly. Following chitosan treatment, fibroblasts reseed on TCPS show lower SA β-gal activity, and higher cellular motility and proliferation ability. Cells can form suspending multicellular spheroids on many biomaterials, but only chitosan is capable of delaying senescence of fibroblasts. Therefore, in addition to the structure of multicellular spheroids, chitosan itself should play an important role in delaying fibroblast senescence. In addition to the intracellular TGF-β expression, the extracellular TGF-β expression is also downregulated by chitosan. TGF-β signaling pathway is involved in the chitosan-mediating fibroblast senescence process. Finally, whether the senescence-delaying effect of chitosan can be applied to other cells, such as human synovial membrane derived cells (SCs) and anterior cruciate ligament fibroblasts (ACLs) is examined. From the studied data, we find that chitosan not only delays the senescence but also enhances the functions of SCs and ACLs, which is benefit for chitosan applying on the cell therapy. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:13:00Z (GMT). No. of bitstreams: 1 ntu-106-D02548002-1.pdf: 7471008 bytes, checksum: 04e7679026c47d7461a6fdbe00af506c (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 摘要 I
ABSTRACT IV GRAPHICAL ABSTRACT VI TABLE OF CONTENTS VII LIST OF TABLES X LIST OF FIGURES XI CHAPTER 1 INTRODUCTION 1 1.1. Background 1 1.2. Purposes 2 1.3. Experimental Design 3 CHAPTER 2 LITERATURE REVIEW 4 2.1. Chitosan 4 2.2. Three Dimensional (3D) Culture for in vitro Tumor Models 5 2.3. Coculture System for in vitro Tumor Models 6 2.4. CD44 in Mesenchymal Stem Cells and Cancer Cells 7 2.5. Senescence 8 2.6. Senescence on Human Synovial Membrane Derived Cells and Anterior Cruciate Ligament Fibroblasts 10 CHAPTER 3 MATERIALS AND METHODS 11 3.1. Biomaterial Preparation 11 3.2. Material Identification 12 3.3. Cells 12 3.4. Cell Viability (mitochondria activity) 16 3.5. Surface Protein Expression 17 3.6. Intracellular Protein Expression 17 3.7. Senescence-associated β-galactosidase (SA β-gal) 18 3.8. Cell Survival 19 3.9. Proliferation Ability 19 3.10. Mobility 20 3.11. Cell sorting with the size and auto-fluorescence 20 3.12. Extracellular Protein Expression 20 3.13. Safranin O Staining 21 3.14. Statistical Analysis 21 CHAPTER 4 RESULTS 22 4.1. Coculture System: Core/shell Multicellular Spheroids as an in vitro Tumor Model 22 4.1.1. Basic Identification of Chitosan Coating TCPS 22 4.1.2. Basic Characteristic of Cells Cultured on Chitosan 23 4.1.3. CD44 Expression of Cells Cultured on Chitosan 23 4.1.4. Cadherin Expression of Cells 26 4.1.5. Cell Distribution of Coculture Cells Cultured on Chitosan 26 4.1.6. The effect of calcium-dependent adhesion molecules on cell distribution 27 4.1.7. Cell distribution of multicellular spheroids on PVA and polyHEMA 29 4.1.8. Cell Mitochondria Activity and Drug Resistance of the in vitro Tumor Model 29 4.2. (Monoculture) Delaying the Senescence of Primary Human Fibroblasts 33 4.2.1. Senescence-like Changes in Human Foreskin Fibroblasts 33 4.2.2. Chitosan Treatment to Senescent Human Foreskin Fibroblasts 34 4.2.3. The Effect of Chitosan Treatment on Senescent Human Foreskin Fibroblasts 34 4.2.4. Effect of chitosan treatment on different cell population 36 4.2.5. PVA and pHEMA Treatment to Senescent Human Foreskin Fibroblasts 37 4.2.6. Senescence-related Protein Expression of Fibroblasts after Biomaterial Treatment 38 4.2.7. Human Foreskin Fibroblasts Cultured in Chitosan- containing Cultured Medium 39 4.2.8. Interaction between chitosan and TGF-β 40 4.2.9. Senescence-like Changes in Human Synovial Membrane Derived Cells and Anterior Cruciate Ligament Fibroblasts 42 4.2.10. The Effect of Chitosan Treatment on Senescent Human Synovial Membrane Derived Cells and Anterior Cruciate Ligament Fibroblasts 43 CHAPTER 5 DISCUSSIONS 45 5.1. Coculture System: Core/shell Multicellular Spheroids as an in vitro Tumor Model 45 5.1.1 The CD44 expression of Cells Cultured on Chitosan 45 5.1.2 Cell Distribution of Coculture Cells Cultured on Chitosan 48 5.1.3 Cell Mitochondria activity and Drug Resistance of the in vitro Tumor Model 52 5.2. (Monoculture) Delaying the Senescence of Primary Human Fibroblasts 55 5.2.1 Chitosan Treatment on Senescent Human Foreskin Fibroblasts 55 5.2.2 Chitosan Delaying the Fibroblast Senescence through TGF-β Pathway 58 5.2.3 Chitosan Treatment on Human Synovial Membrane Derived Cells and Anterior Cruciate Ligament Fibroblasts 63 CHAPTER 6 CONCLUSIONS 67 REFERENCE 68 TABLES 82 FIGURES 85 APPENDIX EDUCATION AND PUBLICATION LIST 114 | |
| dc.language.iso | en | |
| dc.subject | 纖維母細胞 | zh_TW |
| dc.subject | 幾丁聚醣 | zh_TW |
| dc.subject | 核殼共培養多細胞球 | zh_TW |
| dc.subject | 腫瘤模型 | zh_TW |
| dc.subject | 老化 | zh_TW |
| dc.subject | 轉化生長因子-β | zh_TW |
| dc.subject | fibroblasts | en |
| dc.subject | TGF-β | en |
| dc.subject | senescence | en |
| dc.subject | tumor models | en |
| dc.subject | core/shell coculture multicellular spheroids | en |
| dc.subject | chitosan | en |
| dc.title | 幾丁聚醣於癌細胞共培養系統與纖維母細胞老化延遲之影響 | zh_TW |
| dc.title | The Effect of Chitosan Treatment on Coculture System of Cancer Cells and Senescence-Delaying of Fibroblasts | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王至弘(Jyh-Horng Wang),邱信程,洪士杰,林宏殷,李亦淇 | |
| dc.subject.keyword | 幾丁聚醣,核殼共培養多細胞球,腫瘤模型,老化,纖維母細胞,轉化生長因子-β, | zh_TW |
| dc.subject.keyword | chitosan,core/shell coculture multicellular spheroids,tumor models,senescence,fibroblasts,TGF-β, | en |
| dc.relation.page | 115 | |
| dc.identifier.doi | 10.6342/NTU201704456 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-12-12 | |
| 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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