利用去細胞豬角膜為載體並培養兔子上皮與內皮細胞重建全層角膜之角膜再生研究

Abstract

根據世界衛生組織(WHO)統計，1990年全球失明人口為3800萬，於1996年達4500萬，推估到了2020年時，將有多達7600萬人。角膜疾病或傷害造成眼睛失明影響全世界約千萬人口，且未來人口年齡結構老化趨勢，需要眼角膜的病患增加，在捐贈角膜來源有限、而受捐贈者的病情日益複雜的情況下，尋找取代角膜移植的方法也愈形急迫，於是利用組織工程技術來開發人類眼角膜顯得相當重要。有研究顯示出豬的眼角膜於生理功能與解剖構造皆與人的相似，相當適合作為人類眼角膜的替代基質，並運用在層狀角膜移植手術或是基質層替代物。 本實驗目的在於利用去細胞之豬角膜(acellular porcine corneal matrix, APCM)作為載體，培養兔子角膜細胞於上，建構完整的全層角膜替代物。研究中嘗試利用化學性與酵素法來去除豬角膜內之細胞，結果顯示我們能再在完全除去原本之細胞外，還留有大部分的基質與適當的孔洞，製造適合讓細胞生長於內的為環境。接著，本實驗設計簡單模型，利用環形塑膠物製造出隔間，使得細胞能在獨立的環境下生長，目前使用兔子的角膜緣幹細胞培養於角膜上層，分化成上皮細胞，藉由螢光顯微鏡可以看到完整排列整齊的佈滿上層；中間培養基質細胞；內皮層培養兔子的內皮細胞，發現細胞也能佈滿下層，因此本實驗建構之方法能有效讓三種不同的細胞長於角膜替代物上。期望未來能用此新興材料解決捐贈角膜不敷使用的窘境。

According to the World Health Organization (WHO) statistics, the population of the blind approached 55 million in 1990, up to 45 million in 1996. Estimated that by 2020, there will be as many as 76 million people. Corneal disease or injury affects, even resulted in blindness, the world's population about 10 million people and the need of donated cornea for patients increases rapidly due to the aging society of population. However, the number of donated cornea is limited, along with the donor's increasingly complex circumstances, looking for a method of replacing corneal transplantation is increasingly urgent. Thus, developing human corneal substitutes with tissue engineering technology is more and more important. The aim of this study is to develop a full-thickness substitute of human cornea. First, acellular porcine corneal matrix (APCM) was chose to be an appropriate natural basal material. Next, the limbal stem cells were reseeded in the decellularized corneal epithelium, differentiating into epithelial cells; keratocytes were cultured in the corneal stromal layer; endothelial cells were cultured in the bottom layer of the decellularized porcine corneal matrix. Experimental result demonstrates that different types of corneal cells have been successfully reseeded and cultured on APCM, building a complete full-thickness corneal replacement. It is promising and this novel corneal substitute may be able to solve the dilemma of donated cornea.