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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 | Tsung-Lin Yang | en |
dc.contributor.author | 楊宗霖 | zh_TW |
dc.date.accessioned | 2021-06-15T00:35:04Z | - |
dc.date.available | 2014-01-20 | |
dc.date.copyright | 2009-01-20 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2009-01-05 | |
dc.identifier.citation | References
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41867 | - |
dc.description.abstract | 唾液腺是人體分泌腺的一種,其主要的功能在掌管唾液的分泌。分泌的唾液可幫助吞嚥和消化食物,也可幫助口腔健康的維護,具有殺菌、增強免疫能力、及減少蛀牙的功能。因此,若失去唾液腺功能,在生活品質上以及生理健康方面,均會受到相當大的影響。唾液腺功能的損失,可由唾液腺本身的疾病所造成,也可因接受醫療而導致,如頭頸部癌症的患者。當這類病患接受手術與後續的化學和放射線治療,唾液腺的功能會受到嚴重影響。這些病人因無充足的唾液分泌,常常會有口乾的症狀,因而伴隨生活上許多不便和健康的危害。然而,目前在臨床上,對於唾液腺功能缺損的症狀並無根本的治療方法。許多現有的治療方法多只針對症狀的暫時緩解,故很少能藉此得到口乾症狀的舒緩。有鑑於此,如何利用組織工程的技術,重現體內自然發育時唾液腺生成的過程,為重建唾液腺功能最理想的解決方式。
唾液腺的發育起源於口腔的黏膜上皮。在組織生成初期,特定的口腔上皮層會進行組織特化。由於唾液腺為分枝之分泌管腺,故在發育過程中,分枝形態的建立是重要的階段。藉此過程,唾液腺得以利用有限的細胞數,在有限的生長空間中,發育出大量具有功能的唾液腺組織,以滿足體內生理上的需求。此外,這個分枝發育的過程也確立了唾液腺分泌唾腺的運送管道。經由精細連結的管道網路,使產生的大量唾液腺得以順暢地排至口腔,發揮唾液的功能。而在形態建立後的唾液腺發育,特化的唾液腺上皮層會進行分化,進而發育成完整的唾液腺體。 過去對於唾液腺的組織工程的研究,大多侷限於唾液腺的細胞層次。對具有複雜組織構造的唾液腺,這樣的組織工程技術尚未提供真正有效的解決方法。在這些具有複雜組織構造的分泌腺體,他們的功能大半決定於組織結構的形成,而非由單獨的細胞作用即可完成。故對於這些器官的再生,組織工程的研究必須去考量組織間結構形態的生成,及不同組織和細胞間的交互作用。然而,過去對於唾液腺的組織和其結構的再生,並無任何研究提出相關的進展。因此,在本研究中,我們採取組織工程的方法,去探討是否有適合的生醫材料,可用於體外培養系統中,重現或是促進唾液腺組織結構的生成。首先,我們觀察不同生醫材質對於唾液腺組織的影響。我們發現,唾液腺的上皮和間葉組織,其本身的組織特性會受培養時接觸的生醫材質影響。在適當的材料上,不但組織可維持其生物特性,而且組織間的交互作用亦可被引發,而促使形態生長分子和基質分泌,進而促進組織形態的發生。根據這樣的結果,我們進一步採取大規模的篩檢,藉由之前發現的一些材料特性和原則,去尋找最適當唾液腺發育的材料。對於唾液腺的組織,不同的生醫材料展現不同的特性,有些不但不會促使組織的交互作用,甚至影響組織的存活,我們嘗試過的材質包括人工合成或天然的,生物可分解性或非分解性的,以及生物來源的高分子。我們的結果顯示,具有幾丁聚醣材質的培養環境具有促使唾液腺組織形態發育的特質。 我們嘗試先用薄膜培養系統來觀察唾液腺組織的生成情形。在多方的嘗試下,幾丁聚醣所形成的薄膜對於唾液腺組織結構的再生有助益。唾液腺組織培養在這種系統的薄膜之上,可維持其原來的生存能力,並可增生及進行結構發育,而其效率遠大於其他的生醫材料。在詳細的機轉探討中發現,幾丁聚醣的薄膜可提供唾液腺組織一個良好的生存環境,這樣的組織支架可和培養的組織間產生互動,促使培養的組織分泌結構生成和形態發育所需的細胞外基質和相關分子,而且分泌出的細胞外基質可排列成組織在形態發育過程所需的特殊形態。除此之外,我們又研發出另一含幾丁聚醣的唾液腺組織培養系統,而使生醫材料形成的組織支架混合於組織培養液中。在這個方法中,我們發現唾液腺的結構,如分枝發育的形態,可在培養系統中,得到有效率的提升。在幾丁聚醣的培養系統上,這些和唾液腺組織互動所產生的形態決定因子仍保有其活性,可促使後續培養於上之唾液腺組織發育出相關的組織結構,而重現胚胎發育器官形成的過程。 此外,針對聚丁聚醣在唾液腺分枝結構形成的特異性,我們也進而加以比較。我們的結果發現,形態發生的效果和幾丁聚醣的濃度成正比,而且相似的化學結構物並不能重現幾丁聚醣的性質。而幾丁聚醣的單體也無法呈現這樣的效果,且高分子結構也非唯一的因素。然而,這樣的形態生成效果和聚丁聚醣的分子量有直接相關。當幾丁聚醣的高分子鍵結被打斷,形態發生的效果就會消失,這樣的結果告訴了我們有關幾丁聚醣的特異性和應用於組織形態再生的條件。 唾液腺發育主要源自於唾液上皮細胞。在我們的系統中,一些已知的唾液腺上皮的形態決定因子,如纖維生長因子及肝素生長因子的功能均會被加強。這些因子不只在細胞增生的能力被提升,而且組織移動和趨化的能力也被向上調控,而得以促使唾液腺上皮更有效率地針對這些形態因子的調控加以反應。由於這樣含幾丁聚醣的唾液腺組織培養系統不含血清,因此可降低血清的製備和生物相容性的問題,減少了未來直接應用於臨床的阻礙。而且混合培養液的生醫材料,可使相關的培養條件不受空間的限制,增加其應用的可能性。這樣的結果提出了對構成組織支架的生醫材料一個新的發現,也首度利用組織工程的方法去重現唾液腺結構的生成。由於這樣的系統可更有效率的增進形態決定因子和生長因子的功能,使得往後的臨床應用問題得以解決,進而縮小實驗室成果與臨床運用、甚至規模量產的差距。 唾液腺組織的再生和重建,是現今再生醫學和組織工程的重大挑戰。唾液腺是個主要負責唾液生產和調控的器官,其複雜的分枝結構形成了一個有效率的分泌管道。在這個系統中,每個負責細胞各司其職,藉由精密串連的管路,準確而足量地將唾液送至我們的口腔和上消化道去執行功能。我們的研究提出了對於模擬這些精細管路結構生成,首度有了組織工程的初步方法。然而利用組織工程的概念去重建細胞和組織功能,是一個重要但又相當複雜的研究方向。吾人期待藉由類似組織構造層面的研究突破,使得具有完整的結構和功能的組織器官可以生成。如此對於複雜器官再生醫學的進步,及利用組織工程技術重建人類器官的夢想,將踏出了邁向實現的一大步。 | zh_TW |
dc.description.abstract | Salivary gland is an exocrine gland that is responsible for saliva production, absorption, and regulation. In histology, salivary is a ramified tissue formed by interconnecting branches and ducts. The arborized architecture, which is formed by the developmental process of branching morphogenesis, is essential for salivary function. Branching morphogenesis is an efficient and ubiquitous process for creating a larger cellular area for metabolic requirement in developing many glandular organs. To regenerate the glandular organ, such as salivary glands, recapitulation of branching processes may be requisite. At present, though with the progress in preserving phenotypes and promoting differentiation of salivary cells for regenerative purpose, to facilitate the morphogenesis of salivary tissue by tissue-engineering approaches has never been thoroughly explored. In this study, the possibility of promoting salivary gland morphogenesis is explored by tissue-engineering approach step by step, and the way that the chitosan-based biomaterial affects salivary tissue morphogenesis is characterized.
For the purpose of recapitulating salivary gland morphogenesis, the interaction between epithelia and mesenchyme is required. During the development of ectodermal organ, the epithelium interacts with the surrounding mesenchyme to form specific phenotypes by receiving the guiding morphogenetic information. We first use murine fetal submandibular gland (SMG) model and the biomaterials which had been explored for salivary cells regeneration to study the biomaterial effects on epithelial-mesenchymal interaction and salivary morphogenesis. It is found that viability, migratory ability, and tissue interaction of salivary tissue are largely affected by different biomaterials. When salivary tissue is cultured on an appropriate substratum, the epithelial-mesenchymal interaction and the tissue-specific morphogenesis could be induced. Next, we perform global screening to find out the best cultured biomaterial which is capable of promoting morphogenesis of salivary tissue. It shows that the biomaterial effects still exist when whole salivary progenitor tissues are used in the survey. Numerous biomaterials, including synthetic, natural, biodegradable, non-biodegradable, and biological-origin polymers have been investigated. It is found that the cell behaviors as well as the tissue morphogenesis of salivary origin are biomaterial-dependent. Among them, chitosan shows a superior morphogenesis-promoting capacity, which maintains tissue viability and promotes an appropriate tissue interaction for morphogenesis. When chitosan is prepared in the membranous form, it is capable of providing a more preferential environment for salivary gland branch formation. After culturing SMG explants on chitosan membranes, secreted extracellular matrices distribute in a reticular manner and form thicker fibers beyond the extents of cell attachment, which are not found in other biomaterials. In addition, the conditioned chitosan membranes are able to further enhance SMG branching. The fact that the promoting effects are eliminated with collagenase treatment and that type I and III collagen are identified within the adherent fibrillar extracellular matrix raise the possibility that the stimulating factors are collagen-originated. Furthermore, when chitosan is prepared in a soluble form, the morphogenesis-promoting effects are also observed. This result indicates that chitosan is a bioactive substratum for salivary tissue morphogenesis which enables active interaction between cultured salivary tissue and biomaterial. Next, the specificity of chitosan’s morphogenesis-promoting effects is further investigated. It is found that chitosan is able to promote SMG branching in a dose-dependent manner. The effect is chitosan-specific and is not reproduced by substrates with similar chemical structures or by other polymeric molecules of natural or synthetic origin. Furthermore, the branch-promoting effect is molecular weight-dependent. In addition, following digestion with lysozyme, chitinase, or chitosanase, digested chitosan is unable to reproduce the similar effects. This study clarifies the specificity and preferential activity of chitosan in enhancing branching morphogenesis of progenitor salivary tissue. With chitosan, the morphogenesis-promoting effects of mesenchymal tissue on SMG are further enhanced. Chitosan is also competent to induce recombined SMG epithelium to form branches in the serum-free condition. In the presence of chitosan, the morphogenetic efficacy of mesenchyme-derived growth factors responsible for epithelial morphogenesis increases. The specific epithelial phenotype induced by individual growth factor is promoted by chitosan as well. Moreover, the proliferative and the chemotactic properties of these growth factors toward SMG epithelia are also reinforced by chitosan. Therefore, in orchestrating and intensifying the essential mesenchyme-derived growth factors, chitosan is versatile in mediating SMG epithelium to form a predetermined phenotype more efficiently and comprehensively. In all, the current study demonstrates that the morphogenesis of salivary tissue could be regulated by tissue-engineering approaches. It is suggested that, for salivary tissue, chitosan is a morphogenesis-regulating biomaterial. We design a novel methodology to facilitate salivary tissue morphogenesis by enhancing branch formation. The results provide a novel insight into the role of chitosan in salivary tissue morphogenesis and highlight the potential for future application in salivary tissue investigation and regeneration. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T00:35:04Z (GMT). No. of bitstreams: 1 ntu-97-D92444003-1.pdf: 5497123 bytes, checksum: 73553426d527c40608c4b097aae3468b (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 中文摘要 1
Abstract 6 Chapter 1. Introduction 10 1-1. Salivary gland 10 1-2. Xerostomia and treatment of salivary gland dysfunction 12 1-3. Branching morphogenesis and salivary gland development 13 1-4. Tissue engineering of salivary gland 15 1-5. Aims and study design of the dissertation 16 Chapter 2. The effect of biomaterials on epithelial-mesenchymal interaction and morphogenesis of progenitor salivary tissue 19 2-1. Introduction 19 2-2. Materials and Methods 22 2-2-1. Preparation and characterization of biomaterials 22 2-2-2. Culture of isolated salivary epithelia and mesenchyme 22 2-2-3. 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) assay 23 2-2-4. Scanning electron microscopy 24 2-2-5. Salivary tissue recombination assay 24 2-2-6. Immunohistochemistry of type III collagen expression 25 2-2-7. Immunofluorescence of collagen expression and basement membrane formation 26 2-3. Results 28 2-3-1. Characterization of membranes 28 2-3-2. The biomaterial effects on the isolated salivary epithelia and mesenchyme 28 2-3-3. The interaction between biomaterial surface and salivary tissue 32 2-3-4. Type III collagen expression in the salivary epithelial-mesenchymal interface 34 2-3-5. De novo basement membrane synthesis in the salivary epithelial-mesenchymal interface 36 2-3-6. The morphogenesis of salivary tissue recombinants on biomaterials 38 2-4. Discussion 40 Chapter 3. Screening of biomaterials for branch enhancement of salivary gland 45 3-1. Introduction 45 3-2. Materials and Methods 47 3-2-1. Preparation and characterization of membranes 47 3-2-2. Organotypic culture of salivary gland 47 3-2-3. Degradation test 48 3-2-4. 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) assay 49 3-2-5. Immunohistochemistry of collagen deposition 49 3-3. Results 51 3-3-1. Salivary gland morphogenesis on different biomaterials 51 3-3-2. Substrates degradation and associated effects on salivary gland morphogenesis 56 3-3-3. Salivary gland viability on different biodegradable biomaterials 58 3-3-4. Cell migration of salivary cells on biomaterials 60 3-3-5. Deposition of extracellular matrix of salivary gland on different biomaterials 62 3-4. Discussion 64 Chapter 4. The effects of chitosan on salivary gland branching morphogenesis 68 4-1. Introduction 68 4-2. Materials and Methods 70 4-2-1. Preparation and characterization of membranes 70 4-2-2. Medium and Reagents 70 4-2-3. Ex vivo organ culture of SMG explants 71 4-2-4. Scanning electron microscopy 72 4-2-5. Preparation and culture of SMG explants on conditioned membranes 72 4-2-6. Immunohistochemistry and quantification of collagen deposition 73 4-3. Results 75 4-3-1. Branching morphogenesis of SMG explants on PVA, chitosan and PC membranes 75 4-3-2. Extracellular matrix deposition on substrates after cultured with SMG explants 77 4-3-3. SMG branching on conditioned membranes 79 4-3-4. SMG branching on conditioned membranes treated with collagenase 81 4-3-5. Expression and quantification of collagen on conditioned membranes 83 4-3-6. Soluble chitosan promotes branching morphogenesis of SMG explants 86 4-4. Discussion 88 Chapter 5. The specificity of chitosan in promoting branching morphogenesis of salivary gland 93 5-1. Introduction 93 5-2. Materials and Methods 95 5-2-1. SMG ex vivo organ culture 95 5-2-2. Preparation of chitosan-containing culture medium 95 5-2-3. Preparation of chitosan monomers, analogues, and related polymeric substrates 96 5-2-4. Enzyme digestion assay 96 5-3. Results 98 5-3-1. Chitosan promotes SMG branching morphogenesis in a dose-dependent manner 98 5-3-2. Effects of GAGs on SMG branching morphogenesis 100 5-3-3. Effects of chitosan monomers and analogues on SMG branching morphogenesis 101 5-3-4. The effects of natural and synthetic polymers on SMG explant branching 103 5-3-5. The effect of chitosan molecular weight on SMG explants branching 105 5-3-6. The effect of lysozyme, chitinase, and chitosanase on the chitosan-mediating SMG branching morphogenesis 107 5-4. Discussion 112 Chapter 6. Mechanism of chitosan branch-promoting effects in salivary gland morphogenesis 116 6-1. Introduction 116 6-2. Materials and Methods 118 6-2-1. Submandibular glands ex vivo organ culture 118 6-2-2. Antisense oligodeoxynucleotides assay and the rescue experiments with fresh medium 119 6-2-3. Supplement of exogenous branching morphogens 119 6-2-4. Culture of SMG epithelium 120 6-2-5. Mesenchyme recombination assay 121 6-2-6. Cell proliferation assay 121 6-2-7. Chemotactic assay with HGF soaked beads 122 6-3. Results 124 6-3-1. Chitosan effects on the SMG morphogenesis with morphogen down-regulation 124 6-3-2. Synergism of chitosan and exogenous morphogens on SMG morphogenesis 127 6-3-3. The branching-promoting effects of mesenchyme were enhanced by chitosan 130 6-3-4. Chitosan effects on SMG epithelium morphogenesis with homotypic mesenchymal recombination in serum-free culture 132 6-3-5. Chitosan effects on SMG epithelial morphogenesis induced by FGF7, FGF10, and HGF 135 6-3-6. Chitosan effects on SMG cell proliferation induced by FGF7, FGF10, and HGF 140 6-3-7. The chemotactic capacity of HGF beads with chitosan 142 6-4. Discussion 145 Chapter 7. Conclusion and Perspective 150 7-1. Conclusion 150 7-2. Perspective 151 References 153 Appendix 169 | |
dc.language.iso | en | |
dc.title | 幾丁聚醣在唾液腺形態組織工程之研究 | zh_TW |
dc.title | Characterization of Chitosan in Tissue Engineering
of Salivary Gland Morphogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 王盈錦,何弘能,楊怡和,侯勝博,宋信文,楊銘乾 | |
dc.subject.keyword | 幾丁聚醣,唾液腺,形態生成,分枝, | zh_TW |
dc.subject.keyword | Chitosan,Salivary gland,Morphogenesis,Branching, | en |
dc.relation.page | 172 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-01-05 | |
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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