| Objective: To research the growing characteristics of amniotic epithelial cells(AECs) in vitro by culturing the mid and late trimester of pregnancy of AECs of rabbits and rats; and to investigate the effect of using either EGF or 10%FBS and the combination of both on the proliferation of AECs; to explore the feasibility of using AECs to re-establish corneal surface layer by planting AECs on fresh corneal stroma and stratificating AECs.Methods: (1) The rabbits and rats AECs in mid and late trimester of pregnancy were cultured respectively by using tissue inoculation or digesting method; the morphologic changes of cultured AECs were observed by HE staining, inverted microscope and scanning electron microscope; the expression of ck3/12 in AECs was detected by using immunohistochemical staining with cytokeratin monoclonal antibody AE1/AE3 and AE5. (2) AECs were cultured separately by using DMEM/F12 nutrient solution added either 20ng/mlEGF, or 10%FBS, or 20ng/mlEGF and 10%FBS, or neither of them. Then MTT method was used to detect proliferation of AECs in four different nutrient solutions. (3) The cultured AECs of the 2 or 3 generation were planted on fresh corneal stroma, which had been handled with cell adhesion agent. The AECs were stratificated and became corneal-like stratificating epithelial cells by using the air-lifting cultivation that formed by Millicell culture plate inserts. Then, after fixed and dehydrated, the AECs were observed under the scanning electron microscope and the light microscope after HE staining and detected to evaluate the expression of cytokeratin ck3/12 by using immunohistochemical staining with monoclonal antibody AE1/AE3 or AE5.Results: (1) The morphology and growing characteristics of mid and late trimester of pregnancy of AECs of rabbits or rats were similar. After cultured 12h in vitro, the rabbits and rats AECs in the mid and late trimester of pregnancy were splayed... |
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