RESUMO
AIM: To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells. METHODS: A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button. RESULTS: Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane. CONCLUSIONS: Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.
Assuntos
Queimaduras Químicas/cirurgia , Células Epiteliais/química , Epitélio Corneano/química , Queimaduras Oculares/cirurgia , Queratinas/análise , Limbo da Córnea/patologia , Fosfoproteínas/análise , Transativadores/análise , Idoso , Queimaduras Químicas/metabolismo , Células Cultivadas , Transplante de Córnea/métodos , Proteínas de Ligação a DNA , Células Epiteliais/transplante , Epitélio Corneano/transplante , Queimaduras Oculares/metabolismo , Genes Supressores de Tumor , Humanos , Queratina-14 , Queratina-3 , Masculino , Fenótipo , Transplante de Células-Tronco/métodos , Fatores de Transcrição , Transplante Autólogo , Proteínas Supressoras de TumorRESUMO
Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.
