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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64798完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊台鴻 | |
| dc.contributor.author | Shao-Hsuan Chang | en |
| dc.contributor.author | 章韶軒 | zh_TW |
| dc.date.accessioned | 2021-06-16T22:59:35Z | - |
| dc.date.available | 2016-08-10 | |
| dc.date.copyright | 2012-08-10 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-07 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64798 | - |
| dc.description.abstract | 角膜內皮層在角膜中具有相當重要的生理功能，主要為維持角膜的清澈度，並且已知無法於體內再生。由於角膜捐贈的來源短缺大大地限制了角膜移植手術，然而角膜的需求量卻持續增加。再者，非免疫代償性功能失調以及異體移植產生的排斥反應亦可能造成移植手術的失敗。 因此在本研究中，我們發展了一種新方法培養大鼠近端腎小管細胞並以該細胞作為提供角膜內皮來源的替代物。 我們利用幾丁聚醣為培養基材並評估其對近端腎小管細胞的影響，以膠原蛋白培養基材作為對照組。培養在幾丁聚醣上的近端腎小管細胞表現顯著的生長及ERK活化，在第八天達到滿盤的狀態，細胞分化出現並伴隨運輸性上皮的特徵－圓頂化的呈現。細胞圓頂化現象被認為是平行於體內管狀分支主動運輸功能的體外表現。穿上皮電阻的評估顯示培養在幾丁聚醣的近端腎小管細胞具有很高的跨上皮運輸功能。此外，Na+-K+ ATPase 的螢光顯示其位於細胞基底側且完整的表現。而Na+-K+ ATPase 對維持角膜內皮層的功能有重要的影響。近端腎小管細胞層之取得必須連同幾丁聚醣薄膜，薄膜可藉由簡單方法製備而成。細胞層之ZO-1以及Na+-K+ ATPase 的免疫螢光染色幾乎完整表現。掃描式電子顯微觀察顯示細胞層上的細胞呈卵圓狀並具有絨毛。將該細胞層片植入兔子角膜之後表面。然而，異種移植的結果並不能維持角膜基質適當的水合度。我們的結果為近端腎小管細胞提供新的培養方法，以幾丁聚醣作為培養基材對於腎小管功能發育的研究或許會是一個有用的工具，而幾丁聚醣細胞層片對於未來臨床應用亦同樣具有可期待的發展與潛力。
關鍵詞：角膜內皮層；腎小管近端細胞；幾丁聚醣；圓頂化；主動運輸 | zh_TW |
| dc.description.abstract | The corneal endothelium is physiologically the most important monolayer of the cornea and plays a pivotal part on the maintenance of corneal transparency and is not known to regenerate in vivo. Several corneal transplantations have been greatly restricted due to the global shortage of donor corneas, whereas the demands for corneal grafts have been gradually increasing. Furthermore, Non-immunologic endothelial decompensation and allograft endothelial rejection may lead to progressive graft failure. In the present study, we developed a novel method for primary culture of rat proximal tubule cells (PTCs) as an alternative for graft material. We evaluated the effects of chitosan membrane on the primary cultured rat PTCs by comparing with collagen surface. Rat PTCs cultured on chitosan surface displayed a prominent expression of proliferation marker and ERK activation. 8 days after reaching confluence, PTCs showed specific characteristic of transporting epithelia, the formation of domes, which mimic the onset of differentiation. Dome formation is thought to parallel tubular differentiation and morphogenesis in vivo and represents the result of active water transport. The transepithelial electrical resistance evaluation revealed that differentiated PTCs established on chitosan surface exhibited high capacity for transepithelial fluid transport. Moreover, Immunofluorescence revealed that the Na+-K+ ATPase, which is critical for the proper physiological function of the corneal endothelium, was ubiquitously expressed and localized to the cell-to-cell borders. These cultured PTCs were harvested as intact monolayer cell sheets with chitosan film which prepared via simple process. Immunostaining for ZO-1, Na+-K+ ATPase was positive in the cell sheet. Scanning electron microscopy indicated that these cells were primarily cobblestone-shaped morphology with numerous microvilli. Rat PT cell sheets were placed onto the posterior surface of the transplanted rabbit cornea. However, the results of xenograft transplantation showed the fabricated sheets failed of maintaining proper stromal hydration in vivo. Our results implicate that chitosan membrane would be a valuable tool for studying the function and development of proximal tubule cells, as well as supporting the value of harvested cell sheets for clinical applications.
Key word: corneal endothelium, proximal tubule cells, chitosan, dome formation, pump function | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T22:59:35Z (GMT). No. of bitstreams: 1 ntu-101-R99548051-1.pdf: 10919403 bytes, checksum: ceabd801674c368f78994cbdce0eaa2c (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | Figures IV
中文摘要 VIII Abstract IX Chapter 1 Literature review 1 1-1 Corneal endothelium 1 1-2 Fluid transport across leaky epithelia : the role of tight junction 3 1-3 Renal proximal tubule 6 1-4 The formation of domes 9 1-4.1 Dome is the in vitro character of transporting epithelia 9 1-4.2 Molecular mecganism that influence dome formation 11 1-4.3 Dome formation is associated with epithelial differentiation from development to disease 11 1-5 The properties and applications of chitosan 13 Chapter 2 Materials and Methods 2-1 Materials 16 2-2 Instruments 19 2-3 Preparation of solution 21 2-4 Preparation of chitosan-coated plates and chitosan film 22 2-5 Isolation of rat proximal tubules 23 2-6 Dome formation in PTCs 25 2-7 Measurement of transepithelial electrical resistance 25 2-8 Immunofluroescence staining 27 2-9 Western blotting 27 2-10 Scanning electron microscopy 28 2-11 Alkaline phosphatase assay 28 2-12 Transplatation of rat PTC sheets into a rabbit model 29 2-13 Statistical analysis 30 Chapter 3 Results 31 3-1 Morphology and growth of primary cultured PTCs 31 3-2 Expression of Differentiated renal functions 32 3-2.1 The morphology and expression of ZO-1 35 3-2.2 Brush-border enzyme activity. 36 3-2.3 The validation of dome formation and transepithelial electric resistance 37 3-2.4 The expression and localization of alpha-1 subunit Na+-K+ ATPase 39 3-3 The PTC sheets harvesting with chitosan film 40 3-4 Scanning electron microscopy 41 3-5 Evaluation of rat PTCs in reconstructed rabbit cornea receiving a cultured PTC sheet transplant 42 Chapter 4 Discussion 43 Chapter 5 Conclusion 48 References 49 Appendix 58 | |
| dc.language.iso | en | |
| dc.subject | 角膜內皮層 | zh_TW |
| dc.subject | 腎小管近端細胞 | zh_TW |
| dc.subject | 幾丁聚醣 | zh_TW |
| dc.subject | 圓頂化 | zh_TW |
| dc.subject | 主動運輸 | zh_TW |
| dc.subject | corneal endothelium | en |
| dc.subject | proximal tubule cells | en |
| dc.subject | chitosan | en |
| dc.subject | dome formation | en |
| dc.subject | pump function | en |
| dc.title | 評估以腎小管近端上皮細胞作為角膜內皮層之自體移植基材 | zh_TW |
| dc.title | valuation the use of proximal tubule cells as autograft materials for corneal endothelium | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王一中,黃昭淵 | |
| dc.subject.keyword | 角膜內皮層,腎小管近端細胞,幾丁聚醣,圓頂化,主動運輸, | zh_TW |
| dc.subject.keyword | corneal endothelium,proximal tubule cells,chitosan,dome formation,pump function, | en |
| dc.relation.page | 72 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-08 | |
| 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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