Transplantation Of Cultivated Human Amniotic Epithelial Cells For Limbal Stem Cell Deficiency

Objective: Limbal stem cell deficiency is a pathologic state that limbus of cornea or base material is damaged. Limbal stem cells that maintain updating corneal epithelium, suffer nonreversible damage to result in losing compensation,it often leads to the corneal surface by invading conjunctival epithelium cells,ingrowth of fibrous tissue, stromal scarring,and neovascularization,which cause chronic pain and visual loss.The treatment for limbal stem cell deficiency is arduous.Conservative therapy and traditional penetrating or lamellar keratoplasty have poor curative effect.Auto limbal cornea and allo limbal cornea transplantation can restore impaired cornea,prevent invading of new vessels and false pterygium by propagation, differentiation and centrality transmigration of stem cells of limbal basilar part.Effective auto-limbal transplantation does not appear immunological rejection.But it is fit for patients who suffer simple limbal or binoculus local limbal deficiency.There is limitation in clinical application of auto-limbal transplantation. Allo-limbal transplantation does appear immunological rejection which leads to failure.Amniotic epithetical cells are early product of embryonic development.So they probably contain versatility stem cells.Amniotic epithelial cells of conserved amniotic membrane are expired. Amniotic epithelial cells of fresh amniotic membrane are combined with basal membrane tightly.Those cells can not easily educe the function of stem cells.Therefore,amniotic epithetical cells are detached and cultured. That will give a new choice for treating the functional disorder or deficiency of corneal epithelial cells and limbal stem cells.Methods: After anesthetization, one eye of each 18 New Zealand rabbits (2.0–2.5 kg) was alkali burned by lmol/L NaOH after cutting layers of limbus cornea. Human amniotic membrane was obtained at the time of cesarean section. Amniotic epithelium and endothelium were removed by Trypsin-EDTA treatment. Amniotic epithelial cells was obtained from human amniotic membrane, separating chorion, digested by 0.125% typsine at 37℃for 15min.This program is repeated four times. Then collected the cells after centrifugating at 1000rpm for 6min .The cells were inoculated to culture dish that contained DEME after washing them with PBS and cultivate at incubator which contained 5% CO2 at 37℃.Two weeks later, amniotic epithelial cells was inoculated to amniotic membrane. Transplantation operation was performed after two weeks.These 18 rabbits were randomized into three groups:control group,simple denuded amniotic membrane transplantation group and amniotic epithelial cells transplantation group. Transplantion operation was conducted four weeks later after alkali burn. The corneal changes were observed by a slitlamp ,immunohistochemical analysis were performed.Results: Amniotic epithelial cells were adherenced at 2-3 days after seeded. 1×10~6 cells were obtained from amniotic membrane (15×15cm2). Adherenced amniotic epithelial cells took on clumping growth and the cells were round or polygon.The nuclear is comparatively large.There are distinctive vacuole fat drop in endochylema.The cells were passaged per 2~3 weeks. Immunohistochemical analysis showed AE-5 was positive in cornea tissues of amniotic epithelial cells transplantation group.While,AE-5 was weekly expressed in the surface of cornea in the AM group. AE-5 was not expressed on the cornea of control group.Discussion: Amniotic epithelial cells were successfully isolated and cultured. Cultured amniotic epithelial cells can grow on the surface of amniotic membrane. Through burning cornea by lmol/L NaOH after cutting layers of limbus cornea, could establish limbal stem cell deficiency model.Amniotic epithelial cells transplantation decrease corneal conjunctivization after alkaline burn better than simple amniotic stroma transplantation. We believe that transplantation of amniotic epithelial cells represents an effective technique for ocular surface reconstruction inpatients with severe limbal stem cell deficiency.