以纤维蛋白为基质网架的组织工程角膜上皮的构建

目的：应用组织工程技术以纤维蛋白为基质网架、角膜缘干细胞为种子细胞构建组织工程角膜上皮，为角膜上皮移植提供新的移植材料。方法：将20 g•L^-1的纤维蛋白原溶液和含有10 U•mL^-1凝血酶的催化剂溶液混合构成纤维蛋白基质网架，检测支架材料的一般结构和超微结构；取2 mm×2 mm角膜缘组织，胰蛋白酶消化后移至纤维蛋白胶上培养，观察角膜上皮干细胞的生长情况，2周后进行形态学、超微结构和免疫组织化学观察。结果：纤维蛋白基质网架柔软有伸拉性，孔径70～108 μm，平均三维空孔率为70.4%；扫描电镜下呈多层次网络状；接种36 h后，细胞从组织块边缘迁移，7 d后细胞几乎铺满整个培养板孔底，14 d左右细胞与纤维蛋白胶体形成复合植片。透射电镜下观察仍维持上皮细胞特有的超微结构特征。抗细胞角蛋白K3单克隆抗体AE5和抗p63的单克隆抗体4A4免疫组织化学染色均为阳性。 结论：纤维蛋白可作为角膜上皮干细胞移植的良好支架材料，可为组织工程角膜上皮提供新的移植材料。

Reconstruction of fibrin-based tissue engineering corneal epithelium

Objective To reconstruct the fibrin-based tissue engineering corneal epithelium using limbal stem cells as seed cells and provide new grafting material for corneal epithelium transplantation.Methods The fibrin-based frame was reconstructed by mixing 20 m·L-1 fibrinogen solution and 10 U·mL-1 lthrombin and the normal struture and ultrastructure of frame was detected.The limbus corneal tissue(2 mm×2 mm)was obtained,the fibrin-based frame after trypsind egestion.The growth status of the cells was obsewed,and the changes of morphology,ultrastructure and immunohistochemistry were observed after two weeks.Results Fibrin-based frame was soft and had extensibility.Its dimension was 70-108 μm,the average empty porosity was 70.4%.It was multilayer network observed under scanning electron microscope.The cells migrated from the edge of the tissue 36 h after inoculation,single layer cells were formed after 7 d,the cells were combined with fibrin to form gragting implant after 14 d.The cells remained the typical ultrastructure feature of the epithelium observed under transmission electron microscope.The immunohistochemical results of the anti- cytokeratin McAb AE5 and the anti-p63 McAb 4A4 were positive.Conclusion Fibrin is a suitable frame material for corneal epithelium transplantation,provided a new grafting material for tissue engineering corneal epithelium.