以魚鱗膠原蛋白做為角膜內皮細胞層培養載體以做為後彈力層剝除自動角膜內皮移植術的手術移植體

Fish Scale Collagen as Feeder Layer for Corneal Endothelial Cell-sheet for DSAEK Graft

郭芷綝(Tzu-Lin Kuo)

指導教授 ： 林?輝

國立臺灣大學/工學院/醫學工程學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601972

摘要

角膜內皮細胞因為疾病、外傷、手術操作不當等因素導致細胞數量下降，此時角膜內皮層排水功能會失償，進而導致角膜水腫混濁，甚至有失明的可能性。目前角膜內皮層受損的治療方式是以人體捐贈角膜作手術的置換，然而符合捐贈條件的角膜數量在眼庫內一直呈現短缺狀態，因此開發受損角膜內皮治療方式是相當重要且急迫的。本研究使用取自於魚鱗的膠原蛋白作為角膜內皮細胞層培養載體，並且植入前房作角膜內皮細胞層的修復，是一個極具潛力的角膜內皮細胞的治療方式。
在本研究中，經過去細胞、脫鈣、交聯、弧壓後的魚鱗利用螢光顯微鏡、電子顯微鏡、熱重分析儀來評估細胞殘留率、表面結構分析及無機質殘留率。生物相容性的評估則是使用WST-1及LDH分別對細胞活性及細胞毒性作測試。利用電子顯微鏡及ZO-1免疫螢光染色對培養在魚鱗載體上的角膜內皮細胞緊密連接進行觀察。最後將魚鱗載體連同角膜內皮細胞層植入紐西蘭大白兔前房進行體內生物相容性的評估。
結果顯示經過處理後的魚鱗膠原蛋白載體具有高透明度、高含水率及好的生物相容性，並且植入進前房後不會造成免疫反應發生。培養於魚鱗載體上的角膜內皮細胞特徵與正常角膜內皮相似。因此根據本研究的結果，魚鱗膠原蛋白符合當作角膜內皮細胞層載體的一切條件。

關鍵字

後彈力層剝除自動角膜內皮移植術 ； 角膜 ； 魚鱗 ； 細胞載體 ； 膠原蛋白

並列摘要

Corneal endothelial cell quantity may decline due to diseases, trauma and inappropriate surgery method and this situation will lead to corneal edema and have the possibility for patients to become blind. Nowadays, donor corneas are used to treat endothelial dysfunctions and replace damaged tissue. However, the shortage of donor corneas which can meet the conditions of the donation is always a serious problem. Therefore, developing a new corneal endothelial treatment method is important and urgent. We use collagen membrane derived from fish scale, which is a material with high potentiality, to act as a carrier for corneal endothelial cells and to implant into anterior chamber to treat the damaged endothelium.
In this study, de-cellularized, de-calcificated, cross-linking and curvature fabricated fish scale was characterized by fluorescent microscope for de-cellularized evaluation, scanning electron microscope (SEM) for microstructure examination and thermal gravitation analysis (TGA) for de-calcificated analysis. Biocompatibility was analyzed by WST-1 assay for cell viability and LDH assay for cell cytotoxicity. Besides, SEM and ZO-1 immunofluorescence were used to examine cell junction of the endothelial cells on collagen membrane. Finally, collagen membrane with endothelium layer was implanted into rabbit anterior chamber for evaluation of biocompatibility in vivo.
The results show that the prepared fish scale collagen membrane has advantages of high transparency, water content and excellent biocompatibility and no immune reactions are seen in rabbit anterior chamber after implantation. In addition, the cultivated endothelial cells on the collagen membrane are similar to normal cells in vivo. According to this study, fish scale collagen membrane meets the criteria to serve as a carrier for corneal endothelial cells.

並列關鍵字

DSAEK ； cornea ； fish scale ； cell carrier ； collagen

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