臺灣博碩士論文加值系統

角膜是眼睛前端的一層透明薄膜，若損傷導致角膜出現疤痕或混濁，會造成視力模糊，甚至失明，雖然角膜移植手術為常見的角膜外科手術，也是成功率最高的器官移植手術，但是目前面臨的困境是捐贈角膜供不應求的問題，因此，現階段目標是希望利用組織工程技術開發人類眼角膜替代物，以提供穩定的角膜來源。此外，角膜重建的研究上目前都以組織切片的方法來分析評估，但僅能得到縱切面的影像，若能利用非侵入式的FF-OCT技術取得高解析度的二維組織影像，對於角膜替代物的開發能提供有效且快速的分析。
本研究的目的在於利用去細胞之豬角膜作為支架，將兔子角膜細胞共培養於上，建構一全層的角膜替代物，並使用FF-OCT技術對重建的角膜替代物進行縱切面與橫切面的影像分析，以快速且高解析的方式取得角膜組織的二維結構。
實驗結果顯示，體外培養之兔子角膜上皮與內皮細胞可以成功地共培養在去細胞豬角膜支架上，並形成完整的細胞層。藉由H&E染色可以看到，上皮細胞層因為受到擠壓且脫落，大約保留有3-4層的上皮細胞層，而內皮層一樣有整層脫落的情形，然而，由FF-OCT的影像可以清楚看到，上皮細胞層由5-6層的上皮細胞組成，內皮細胞層則為單層的結構，相較於H&E染色的切片圖，FF-OCT技術較能有效地取得高解析度的角膜重建分析結果。

The cornea is a transparent film in the front of the eye. Diseases of the cornea may lead to corneal opacification, visual impairment and even blindness. Corneal transplant surgery is a common corneal surgery, but also the high rate of success in all organ transplantations. However, there is a severe shortage of suitable cornea donors in many countries with an increased use of the old-aged population and corrective laser surgery. Therefore, the aim of this study was to construct a corneal replacement using acellular porcine corneal matrix (APCM) as a scaffold and coculturing rabbit epithelial and endothelial cells. In addition, studies of corneal reconstruction are currently analyzed and evaluated using tissue slices, but the process of tissue sectioning is quite complex and the processing time is lengthy. If non-invasive FF-OCT technology can quickly obtain biological tissue images and provide high-resolution images, it will be a great contribution in the field of research of artificial cornea.
The aim of this study was to analyze the coculture of rabbit corneal epithelium and endothelium on acellular porcine cornea matrix(APCM) with full-field optical coherence tomography(FF-OCT). Results show that rabbit corneal epithelial and endothelial cells can be cocultured on APCM scaffold in vitro. According to H&E staining, epithelium layer was squeezed and detached, and so did endothelium layer. However, FF-OCT images clearly show that the corneal epithelium was composed of 5-6 layers of cells, and the corneal endothelium was a monolayer. Collectively, the study demonstrate that FF-OCT technology can quickly and efficiently achieve high-resolution analysis results of reconstructed corneal tissue.

中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 vi
表目錄 viii
第一章 緒論 1
1.1 眼角膜構造與功能 1
1.1.1 眼角膜移植 4
1.1.2 眼角膜之移植手術(角膜捐贈) 4
1.1.3 角膜替代物(人工角膜) 6
1.2 再生醫學 9
1.2.1 眼角膜組織工程 10
1.2.2 去細胞豬角膜 11
1.3 OCT在眼角膜之應用 12
1.3.1 OCT發明的起源 13
1.3.2 OCT技術原理 14
1.3.3 OCT的發展歷史 15
第二章 研究概述 16
2.1 研究背景 16
2.2 研究動機與目的 16
2.3 研究架構圖 19
第三章 實驗材料與方法 20
3.1 實驗藥品 20
3.2 實驗儀器 22
3.3 去細胞豬角膜基質(acellular)支架之製備 23
3.4 冷凍乾燥法 24
3.5 DNA含量測定 25
3.6 葡萄糖胺聚糖(Glycosaminoglycan, GAGs)含量測定 26
3.7 掃描式電子顯微鏡(SEM) 27
3.8 光學同調斷層掃描(Optical Coherence Tomography; OCT) 28
3.9 組織切片前處理 28
3.10 利用DAPI螢光染色觀察豬角膜基質支架的去細胞情形 29
3.11 免疫細胞化學染色(Immunocytochemistry; ICC) 29
3.11.1 Cytokeratin 3+12 30
3.11.2 N-Cadherin 30
3.12 角膜初代細胞之培養 31
3.12.1 兔子角膜上皮細胞培養 31
3.12.2 兔子角膜內皮細胞培養 31
3.12.3 牛角膜內皮細胞培養 32
3.13 角膜重建之模型建立 33
3.14 PKH26與PKH67染色 34
3.15 細胞毒性試驗(LIVE/DEAD cell viability assay) 35
第四章 結果與討論 36
4.1 去細胞豬角膜(APCM)之製備與觀察 36
4.2 角膜基質支架的去細胞程度評估 37
4.3 以OCT評估角膜基質支架的去細胞程度 38
4.4 角膜基質支架的結構與性質分析 41
4.5 組織培養之角膜細胞型態觀察 43
4.6 消化法之角膜內皮細胞型態觀察 45
4.7 角膜細胞之免疫螢光染色標定 47
4.8 角膜基質支架對於角膜細胞的毒性評估 49
4.9 角膜細胞共培養於角膜基質支架之生長情形 52
4.10 以H&E染色分析角膜細胞共培養於角膜基質支架之生長情形 53
4.11 以OCT分析角膜細胞共培養於角膜基質支架之生長情形 54
4.12 以OCT分析角膜上皮細胞於氣液介面培養環境下之生長情形 58
4.13 角膜上皮與內皮細胞共培養於角膜基質支架之免疫螢光染色 60
第五章 結論 61
參考文獻 62

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