AIM: To investigate the plasticity of human amniotic epithelial cells induced to differentiate into corneal epithelial cells and the feasibility of corneal epithelium reconstruction . METHODS: A piece of amniotic membrane taken from an uncomplicated elective caesarean section was digested by collagenase , trypsin and EDTA. The amniotic epithelial cells were primarily cultured and passaged in Dulbecco 's modified Eagle's medium containing 10% fetal bovine serum. The HAECs of the 2nd or 3rd generation were continuously cultured on fresh corneal stroma . One or two days later , the human amniotic epithelial cells formed a monolayer , and then the air - lifting cultivation was carried out in an insert culture dish. The cultured cells on corneal stroma were investigated morphologically by hematoxylin - eosin staining, and its ultramicrostructure was studied by scanning electron microscopy ( SEM) and transmission electron microscopy ( TEM). Meanwhile, the expression of cytokeratin ( CK) 3/12 was detected by immunohistochemistry. RESULTS; Human amniotic epithelial cells were successfully reproduced on corneal stroma in vitro. The monolayer was formed after cultured for 1 or 2 days , and stratification with 4 to 5 layers of epithelial cells was observed 14 days after air - lifting cultivation. A wealth of microvilli on the surface of HAECs were observed by SEM , while desmosomes among HAECs were found by TEM . The stratified cells expressed CK3/12. The composed tissue was very similar to normal corneal epithelium . CONCLUSION; Human amniotic epithelial cells can be used as seed cells to reconstruct the corneal epithelium by the technique of tissue engi -neering.%目的:探讨人羊膜上皮细胞(human amniotic epithelial cells,HAECs)诱导分化为角膜上皮细胞的可塑性及其重建角膜上皮的可行性.方法:取足月产人羊膜,通过胶原酶、胰蛋白酶和EDTA消化获得HAECs,将细胞在体外培养扩增、传代,将2～3代HAECs接种于去上皮的兔角膜基质上培养,待HAECs融合形成单层后,置于插入式培养皿中进行气液界面培养14 d.对角膜基质上培养的HAECs用光镜、扫描电镜和透射电镜进行形态学及超微结构观察,以及细胞角蛋白(CK)3/12的免疫组织化学检测.结果:HAECs可在角膜基质上生长,培养1～2 d可形成单层,气液界面培养14 d可形成4～5层细胞的复层上皮,扫描电镜观察细胞表面有丰富的微绒毛,透射电镜下可见上皮细胞之间有桥粒连接,免疫组化检测复层细胞表达CK3/12,所形成的复层结构与正常角膜上皮相似.结论:HAECs有可能作为种子细胞用于组织工程角膜上皮重建.
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