以异种后弹力膜基质为载体培养角膜内皮细胞的研究

Heterogenic Descemet’s membrance/stroma as the carrier for culture of corneal endothelial cells in vitro

目的 探讨以异种后弹力膜 (DM) /基质为载体培养角膜内皮细胞的可行性 ,为培养内皮细胞的临床移植提供新的方法和供体材料。方法 取厚 10 0 μm的猪角膜后层 ,冻存解冻后置中性蛋白酶 3 7℃孵育 5min ,去除内皮细胞 ,保留DM及薄层基质 ,置纯甘油中脱水保存 6个月 ;使用前复水 ,紫外线二面灭菌 ,制备成载体 ;接种兔角膜内皮细胞于载体的DM面并体外培养 ,行细胞形态、密度、组织学、超微结构的观察。结果 兔内皮细胞在猪DM/基质载体上贴附生长 ,形成紧密连接的单层 ,形态近似六角形分布 ;细胞密度达 ( 3 62 0± 110 2 3 )个 /mm2 ,具有正常兔角膜内皮细胞的超微结构。结论 角膜内皮细胞能够在异种DM/基质载体上良好生长 。

Objective To evaluate the possibility of culturing corneal endothelial cells on heterogenic Descemet’s membrane (DM) in vitro. Methods The carrier (DM/posterior stroma) were isolated from porcine corneal posterior lameller.The carrier was frozen and stored at -80℃ and then thawed and incubated with dispase at 37℃ for 5 minutes.Endothelial cells were removed by gentle scraping with a cell scraper,and DM/stroma were preserved in glycerine for 6 months and washed in PBS and sterilized by ultraviolet ray prior to use.Rabbit corneal endothelial cells were seeded onto DM and cultured in vitro,and the morphology,histology,density and ultrastructure of carrier were examined. Results Corneal endothelial cells could attached on heterogenic DM and formed a confluent monolayer of cuboidal-shaped cells containing normal-appearing cytoplasmic organelles with the cell density (3 620±110.23) cells/mm^(2). Conclusion Heterogenic DM/posterior stroma layer is a good carrier for culturing corneal endothelium,which provides a donor material for endothelium cells transplantation.