应用壳聚糖和蚕丝蛋白构建组织工程角膜基质及其移植的实验研究

To construct a scaffold using silk fibroin (SF) and chitosan (CS) that could replace the corneal stroma while remaining the biological characteristics of the scaffold materials. Another goal of this study was to evaluate the potential suitability of an artificial membrane composed of silk fibroin (SF) functionalized by different ratios of chitosan (CS) as a substrate for the stroma of the cornea. Keratocytes were cultured on translucent membranes made of SF and CS with different ratios.To develop an organotypic corneal stroma, SF and CS were chosen to synthesise the tissue engineering bioscaffold. The biophysical properties of the silk fibroin and chitosan (SF/CS) membrane were examined. We cultured primary human corneal epithelial cells , corneal stromal cells, corneal in vitro. The keratocytes were used to assess cytotoxicity on silk fibroin-chitosan (SFCS) blends. The corneal lamellar scaffolds were developed with sequential culture techniques to form cell-scaffold constructs. The resulting cell scaffolds were evaluated using western blot in addition to microscopy. Implantation was tested in animals. The biocompatibility was tested by transplanting the acellular membrane into rabbit corneal stromal pockets. The corneal substitutes were analysed by light and electron microscopy. This study could maintain the biological characteristics of the scaffold materials. It might be a suitable blend for corneal tissue engineering. Taken together, these results indicate that SF/CS holds promise as a substrate for corneal reconstruction.

本课题采用细胞生物学和组织工程学技术，分离、培养、鉴定人原代角膜缘干细胞、角膜上皮细胞、角膜基质细胞和角膜内皮细胞；应用不同比例丝素蛋白和壳聚糖的理化特性合成透明共混膜，对其毒性，体外组织相容性进行检测；体外构建符合解剖学结构的角膜模型，并施行形态学观察；通过对兔的板层角膜移植实验，术后检测移植排斥的各项相关指标，对其体内组织相容性作出评价。本课题将全新材料引入角膜移植领域，为构建组织工程学角膜提供胞外基质网架，也为未来组织工程角膜在临床上的应用提供了实验依据。

目前，世界上拥有大约一千万人正承受着由于角膜损伤或疾病而引起的视力下降。更糟的是，唯一有效地治疗方法就是应用捐献者的角膜进行移植手术。然而，安全且高质量的角膜组织严重短缺，尤其是在第三世界国家和发展中国家。同时，即使手术成功率很高，但术后仍然存在免疫排斥的威胁，主要是针对碱烧伤和首次移植失败的患者。所以，近年来，为了弥补捐献者的缺乏，组织工程角膜发展迅速。本课题的目的就是体外合成重建一种角膜替代物，为角膜移植提供一条新的思路。