脱细胞角膜基质为支架构建组织工程角膜上皮组织的实验研究

Reconstruction tissue-engineered corneal epithelium using xenogeneic acellular corneal stroma as scaffold

目的比较氯化钠（NaCl）-十二烷基硫酸钠（SDS）一胰蛋白酶与分散酶（Dispase）-聚乙二醇辛基苯基醚（Triton-X-100）两种角膜组织脱细胞方法的效果，探讨以脱细胞角膜基质为支架构建组织工程化角膜上皮组织的可行性。方法实验研究。采用完全随机化设计的方法，分别用NaCl-SDS-胰蛋白酶和Dispase—Triton—X-100处理兔角膜组织，使用裂隙灯显微镜、光学显微镜及透射电镜观察经两种方法处理后的角膜基质特性和脱细胞效果。以兔角膜缘上皮细胞为种子细胞，Dispase—Triton—X-100处理的猪角膜前弹力层与基质为支架，体外重建兔角膜上皮细胞层，并进行形态学、组织病理学及免疫组织化学检测。结果两种方法处理过的兔角膜基质大体形态相似，灰白色不透明，水肿明显，质地柔软。组织病理学和超微形态学观察显示两种方法处理的兔角膜基质胶原排列规整，NaCl-SDS-胰蛋白酶处理的角膜组织残留了部分基质细胞碎片，而Dispase—Triton—X-100处理的角膜组织未见基质细胞碎片。兔角膜缘上皮组织块接种于脱细胞猪角膜前弹力层与基质上，24h角膜上皮细胞开始游出，3～4d融合呈片状，7—8d形成单细胞层。组织工程化角膜上皮细胞表达CK3。结论Dispase—Triton—X-100处理方法的角膜组织脱细胞效果良好。以脱细胞猪角膜前弹力层与基质为支架，可在体外构建组织工程化兔角膜上皮组织。

Objective To compare two methods for preparing acellular corneal stroma and evaluate the possibility of culturing corneal epithelium on xenogeneic acellular corneal stroma. Methods Experimental study, applying completely randomized design method. Dispose followed by Triton-X-100 detergent and sodium chloride SDS detergent followed by trypsinase were applied respectively to treat the rabbit cornea. The characteristics of corneal stroma and acellular status after treatment were examined with slit lamp, optical microscope and transmission electron microscope. The rabbit limbal cells were then cultured on the acellular porcine Bowman＇s membrane/stroma. Rabbit corneal epithelium lamella reconstructed in vitro and evaluated in morphology, histopathology and immunohisto-chemistry. Results Acellular corneal stroma prepared by two different methods is quite similar in morphology, being gray and opaque with visible edema and soft texture. Collagen fibers of the stroma were regularly in histopathology and uhrastructure. But the one prepared by the NaCl-SDS-Trypsinase method retained a amounts of cell debris, while there was none in the other did by Dispase-Triton-X-100 method. The limbal cells began to shift out at 24 hours after being inoculated on xenogeneic acellular corneal stroma, then attached on it, formed a confluent monolayer containing normal-appearing in 7 days. The tissue engineering corneal epithelium was cryosectioned and characterized immunohistochemlcally at 14 days after inoculation. Meanwhile, epithelium associated antigen CK3 in endochylema was stained. Conclusions Dispase-Triton-X-100 was proved better in obtaining acellular corneal stroma. It is possible to reconstruction tissue-engineered rabbit＇s corneal epithelium on acellular porcine corneal Bowman＇s membrane/stroma. （Chin J Ophthalmol, 2008,44： 825-830）