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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻 | |
dc.contributor.author | Tsung-Jen Wang | en |
dc.contributor.author | 王宗仁 | zh_TW |
dc.date.accessioned | 2021-06-07T18:12:01Z | - |
dc.date.copyright | 2012-07-16 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-06-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16375 | - |
dc.description.abstract | 角膜內皮細胞是角膜最內層位在角膜間質與前房之間的單一層細胞,平時有維持角膜正常含水量和透明度的功能,角膜內皮細胞若不幸發生外傷性或自發性病變時,會造成病患角膜水腫及混濁進而影響視力。
根據統計,目前世界上至少有超過二千萬以上的病人深受角膜眼盲所苦。角膜移植是治療因角膜疾病而導致失明的唯一方式,以往乃是藉由全層性角膜移植術,這是可能伴隨著眼內大出血、感染、青光眼及高度散光等併發症之手術;同時角膜移植也面臨了器官來源短缺的問題,目前全世界每年只能進行約50,000~60,000例手術。 然而臨床上單純角膜內皮細胞的喪失問題,如偽水晶體水泡角膜病變或是原發角膜內皮細胞退化疾病等,理論上往往只需要置換掉有病變的角膜內皮層, 不一定需要傳統的全層性角膜移植術。目前治療趨勢是進行置換單一角膜內皮層手術來取代傳統的全層性角膜移植術,因其具有快速復原及減少術後併發症發生之優點,但依然需要充分的器官供應得以進行。 角膜移植和其他器官移植一樣,也面臨器官來源短缺的問題,因此以專一性的細胞治療來解決單純角膜內皮細胞的疾病,成為相當熱門的題目,目前趨勢是以人類角膜內皮細胞建立體外培養系統,然而角膜內皮細胞有不易複製的特性,在一般的組織培養皿上容易產生纖維母細胞變性的現象,一旦發生將失去應有的功能,這些問題使得體外製造角膜內皮細胞的研究仍待探討與突破。 細胞行為受到本身內在的因素,以及和其他細胞、訊息及基質之間複雜的交互作用所影響,在組織工程技術中常利用不同的生醫材料提供細胞適合生長的環境來培養細胞,過去研究也發現外在生長因子以及細胞和細胞的交互作用確實會調控角膜內皮細胞的複製與分化,細胞在不同的基質上行為表現也會不同,然而目前不同特性之生醫材料對於角膜內皮細胞的培養仍屬未知,角膜內皮細胞和不同基質之間的交互作用也值得探討。 文獻回顧尚未發現詳細探討生醫材料對於角膜內皮細胞培養的相關研究,在組織工程技術中常利用不同的可裂解或不可裂解生醫材料提供單一種類細胞適合生長的基質來培養細胞,本研究中第一部分首先我們以務實的態度,提出在適當的生醫材料下存在著讓體外培養之角膜內皮細胞複製並維持功能的可行性,利用不同特性之生醫材料做為角膜內皮細胞體外培養的基質,觀察角膜內皮細胞的行為表現,試圖尋找合適讓細胞得以複製並分化以維持其型態與功能的生醫材料。根據第一部分研究結果,以組織工程技術尋找適當生醫材料做為角膜內皮細胞培養基質,能克服其不易複製與容易發生纖維母細胞變性的特點,成功地於體外培養。 此外,以可裂解性生醫材料做為角膜內皮細胞培養的基質,對於動物實驗及日後的實際應用,存在的一定的重要性,藉由此一創新的想法,建立適當的角膜內皮細胞體外培養模式以提供替代來源。第二部份接續過去研究成果,務實提出以混摻兩種可裂解性的生醫材料:幾丁聚醣與聚己內酯,尋找能讓角膜內皮細胞得複製並分化以維持其型態與功能的合適混摻比例,期望於體外形成角膜內皮細胞層。截至目前,文獻回顧尚未發現相關詳細地系統性的研究,相信本研究日後對於解決臨床上的實際問題,存在一定重要性。 | zh_TW |
dc.description.abstract | Corneal endothelial cells (CECs) compose of an intact monolayer in the innermost layer of the cornea and the transparency of the cornea can be sustained only when pump and barrier functions of CECs are maintained. Medically, the corneal endothelium is the most essential part of the cornea, but damages during intraocular surgeries or corneal endothelial diseases such as Fuchs dystrophy can deteriorate the CECs. These events cause corneal edema and opaque which can lead to severe vision impairment, thus corneal transplantation is required to restore cornea clarity and visual acuity.
Conventional penetrating keratoplasty replace the whole corneal layers to treat irreversible opacity. Unfortunately, not only integral structure and optical properties may be altered from a necessary evil of sutures but also higher possibilities of infection and rejection occur. Nevertheless, the majority of corneal transplantations are merely necessary to substitute damaged corneal endothelium. Therefore, supplanting only posterior corneal endothelial layer (endothelial keratoplasty) has numerous benefits including earlier visual recovery, less induced astigmatism and fewer ocular surface complications. Nowadays, the percentage of endothelial keratoplasty increases in the world, but corneal transplantation still faces a global shortage of cornea donors and primary immune rejection. The development of ex vivo culture system by tissue engineering to establish the cultivated corneal endothelial sheet is the current trend. Fortunately, CECs can be cultivated and expanded in vitro and seeded successfully onto natural tissue materials or synthetic polymeric materials as graft for transplantation. There are three key steps involved: isolation, preservation, and expansion. Traditionally, tissue culture plates (TCPS) were used to cultivate corneal endothelial cells. However, the fibroblastic transformation occurs easily. Once this happens, the CECs would lose their functions. To date, still many novel methods are explored continuously. Cell behavior is decided by constitutional programs and complex interactions among cells, signals, and matrix. Biomaterial substrates are usually used to grow cells in tissue engineering. Several reports have shown the usefulness of extrinsic signals from soluble growth factors and cell-cell contact for managing the proliferation and differentiation of corneal endothelial cells. Also, cells may proliferative and differentiate well with suitable biomaterials. Here, the effects of hydrophilic and hydrophobic substrate on corneal endothelial cells are unknown. Recent data had shown possible to find the proper biomaterial for the specific cell in many experiments. And the interaction between cell and matrix is important to be surveyed. Tissue engineering is a new trend in biotechnology using cultured cells and biomaterials to replace damaged tissue and restore impaired functions. After reviewing literatures, we found researching into the behaviors of corneal endothelium on biodegradable polymer membranes had not been inspected well. From our preliminary results, the behavior of corneal endothelial cells would not be the same. Therefore, in the initial part of this study, we try to cultivate corneal endothelial cells on different hydrophilic and hydrophobic polymer membranes. Further efforts to be made are to define the cell-surface interaction among them from investigation of morphology and function. Based on our findings, the appropriate biomaterials can be applied successfully for cultivation of CECs. Eventually, we hope to make clinical application feasible in the future. The goal to fabricate CEC sheet by means of tissue engineering may not be approached by single biomaterial. Hybridizing two polymers is a method to develop novel biomaterials with combinations of properties from individual. Approved by Food and Drug Administration (FDA), chitosan and polycaprolactone (PCL) are biodegradable biomaterials with various advantages respectively. Furthermore, PCL can be introduced into chitosan easily in a harmonic status by the method of blending without complex chemical modifications. Towards this aim, in the subsequent part of this study, blends made from various proportions of biodegradable biomaterials (chitosan and PCL) will be examined in the CEC culture systems to elucidate their possible impact on clinical demand and scientific interest. In this study, we will first hypothesize that it is possible to create a new blended biomaterial that can hybridize the characteristics of chitosan and PCL concurrently to serve as a scaffold and carrier for CEC culture and transplantation. In the future, we hope to provide a model and design transplantation graft for possible clinical applications in tissue engineering and regenerative medicine of the corneal endothelium. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T18:12:01Z (GMT). No. of bitstreams: 1 ntu-101-D97548014-1.pdf: 3730159 bytes, checksum: 88897bf5b123b47a2079a44834a7f132 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Contents
CONTENTS I TABLE V FIGURES VI 摘 要 IX CHAPTER 1 1 BACKGROUND AND LITERATURES REVIEW 1 1.1. MOTIVATION AND OBJECTIVES 1 1.2. CORNEAL ENDOTHELIUM 3 1.2.1. Corneal endothelial cells (CECs) 3 1.2.2. Posterior corneal transplantation 4 1.3. CEC TISSUE ENGINEERING 5 1.3.1. Different polymer membranes for cell culture 5 1.3.2. CEC culture on biodegradable biomaterial 6 1.3.3. Blending of chitosan and PCL 7 1.3.4. Chitosan/ PCL blends 8 1.4. SIGNIFICANCES 10 1.5. REFERENCES 12 CHAPTER 2 14 AN APPROPRIATE BIOMATERIAL FOR PROLIFERATION AND PRESERVATION OF CORNEAL ENDOTHELIAL CELLS IN SUPPLEMENTED HORMONAL EPITHELIAL MEDIUM 14 ABSTRACT 14 2.1. INTRODUCTION 16 2.2. MATERIALS AND METHODS 18 2.2.1. Preparation and characterization of culturing substrates 18 2.2.2. Isolation and culture of BCECs 19 2.2.3. Cell viability assay by Alamar Blue 20 2.2.4. Immunofluorescence study 20 2.2.5. Western blot analysis 21 2.2.6. Quantification of type IV collagen deposition 21 2.2.7. Statistical analysis 22 2.3. RESULTS 23 2.3.1. Contact angle analysis 23 2.3.2. Coating thickness and surface topography 23 2.3.3. Cell adhesion and expansion 23 2.3.4. Cell viability 24 2.3.5. Gross view of corneal endothelial sheet 24 2.3.6. Cell morphology 25 2.3.7. Phenotype expression 25 2.3.8. Type IV collagen production 26 2.3.9. Adsorption of type IV collagen 26 2.4. DISCUSSION 28 2.5. CONCLUSION 32 2.6. REFERENCES 33 CHAPTER 3 38 THE PHENOTYPIC RESPONSE OF BOVINE CORNEAL ENDOTHELIAL CELLS ON CHITOSAN/POLYCAPROLACTONE BLENDS 38 ABSTRACT 38 3.1. INTRODUCTION 40 3.2. MATERIALS AND METHODS 43 3.2.1. Preparation and characterization of culturing substrates 43 3.2.2. Isolation and culture of BCECs 44 3.2.3. Cell adhesiveness 45 3.2.4. Cell proliferation test 45 3.2.5. Morphometric measurements and immunofluorescence microscopy 45 3.2.6. Immunofluorescence study 46 3.2.7. Real-time quantitative polymerase chain reaction (qPCR) 47 3.2.8. Statistical analysis 47 3.3. RESULTS 48 3.3.1. Transparency and transmittance of chitosan/PCL blends 48 3.3.2. Cell adhesion within 4 hours on culturing substrates 48 3.3.3. Proliferation of BCECs 49 3.3.4. Morphometric analysis of BCECs on culturing substrates 49 3.3.5. Cytoskeletal structure of BCECs on culturing substrates 50 3.3.6. Cell Morphology of cultured BCECs 50 3.3.7. Immunofluorescent staining of cultured BCECs 51 3.3.8. mRNA expressions of BCECs on the blends 51 3.4. DISCUSSION 53 3.5. CONCLUSION 57 3.6. REFERENCES 58 CHAPTER 4 63 NOVEL CHITOSAN-POLYCAPROLACTONE BLENDS AS POTENTIAL SCAFFOLD AND CARRIER FOR CORNEAL ENDOTHELIAL TRANSPLANTATION 63 ABSTRACT 63 4.1. INTRODUCTION 65 4.2. MATERIALS AND METHODS 68 4.2.1. Preparation of culturing substrates 68 4.2.2. Measurement of transparency and transmittance 68 4.2.3. Primary culture of BCECs 69 4.2.4. Immunohistochemistry 70 4.2.5. Western blot analysis 70 4.2.6. Preparation of chitosan and PCL blend membranes 71 4.2.7. Seeding of BCECs on PCL 25 membrane 72 4.2.8. Statistical analysis 72 4.3. RESULTS 73 4.3.1. Transparency and transmittance 73 4.3.2. Cell Morphology of cultured BCECs on the culturing substrates 73 4.3.3. Collagen type IV production 74 4.3.4. The macroscopic features of blend membranes 74 4.3.5. Cell morphology and phenotype expression of cultured BCECs on PCL 25 blend membrane 75 4.4. DISSCUSSION 76 4.5. CONCLUSION 80 4.6. REFERENCES 81 CHAPTER 5 CONCLUSIONS AND FUTURE WORK 85 REFERENCES 88 ABBREVIATION 116 LIST OF PUBLICATION 117 LIST OF CONFERENCE 118 | |
dc.language.iso | en | |
dc.title | 生醫材料於眼角膜內皮細胞組織工程之研究 | zh_TW |
dc.title | Application of Biomaterials in Tissue Engineering of Corneal Endothelial Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 胡芳蓉,陳慕師,楊中美,王一中,許紋銘 | |
dc.subject.keyword | 角膜內皮細胞,角膜移植生醫材料,混摻,幾丁聚醣,聚己內酯, | zh_TW |
dc.subject.keyword | corneal endothelial cells (CECs),biomaterials,corneal transplantation,blends, chitosan,polycaprolactone, | en |
dc.relation.page | 108 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-06-28 | |
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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