Primary Study On Human Amniotic Epithelial Cells Being Induced Into Conjunctiva-like Cells In Vitro

Objective:To culture human amniotic epithelial cells and rabbit conjunctiva epithelial cells in vitro,and to investigate the possibility of transdifferentiation of human amniotic epithelial cells to conjunctiva epithelial cells through co-cultrue the two kinds of cells in vitro,thus to find a new kind of seed for tissue engineering conjunctiva epithelium reconstruction,at the same time,to construct artificial conjunctiva epithelial sheet use fibrin gels,thus to lay a foundation for tissue engineering ofconjunctiva epithelium reconstruction.Methods:(1) The mid and late trimester human amniotic epithelial cells and rabbit conjunctiva epithelial cells were cultured respectively using tissue inoculation or digesting methods,the two kinds of cells were observed and identified using inverted microscope, Hematoxylin-eosin staining and cytokeratin immunocytochemistry staining;(2) Use Transwell non-contract co-culture system to co-culture them for two weeks,at the same time add conjunctiva tissue homogenate supematant to the culture fluid.The expression of CK13 and MUC5AC of the pre and post co-cultured epithelial cells were identified using immunofluorescence staining and flow cytometry.(3) After induced,human amniotic epithelial cells were inoculated on fibrin gels sheet,than lift the air-fluid surface to form multilayered epithelial cells,and thus to construct artificial conjunctiva epithelial sheet,10 days after using scanning electron microscopy,HE gaining and immunofluorescence staining to observe and identify.ResuR:(1) The shape and grow feature of human amniotic epithelial cells and conjunctiva epithelium cells cultured in vitro were rather resemble,they were spherical or orbicular-ovate, and after overgrow they were of typical slabstone-like appearance.The conjunctiva epithelial cells cultured by tissue inoculation were tighter arranged and there were a few goblet cells with comparatively large body and abundant endochylema.With HE staining the cells nuclus of them were light blue and the nucleolus were deep blue,they were with plentiful endochylema,fat cell body and clear cell membrane.Cytokeratin monoclonal antibody staining of the cells was positive;(2) After induced culturing for two weeks,CK13 of human amniotic epithelial cells was positive with immunofluorescence staining,but MUC5AC was still negative.Flow cytometry showed CK13 positive cell rate was 75.4%in average and MUC5AC positive cell rate was still less than 1%after inducing.CK13 and MUC5AC of human amniotic epithelial cells were both less than 1%before inducing;(3) Observed with scanning electron microscopy,human amniotic epithelial cells grow on fibrin gels were adhered well,their surface have aplenty microvillus.HE staining displayed human amniotic epithelial cells grow on fibrin gels were formed 2 to 3 layers after lift air-fluid surface cultured for 10 days.Immunofluorescence staining showed that the cytokeratin CK13 of those multilayered cells were positive.Conclusion:(1) Human amniotic epithelial cells and rabbit conjunctiva epithelial cells can be cultured and passaged successfully in vitro;(2) Human anmiotic epithelial cells cultured in vitro can be induced into conjunctiva-like epithelial cells;(3) Lift air surface to culture them with the carrier of fibrin gels,human amniotic epithelial cells sheet can become multilayer and could be expected to reconstruct conjunctival epithelium.