Experimental Study On The Rhesus Monkey Corneal Endothelial Cells Substituted By The Allogeneic Vascular Endothelial Cells Cultivated In Vitro

Objective:It is to investigate the inner surface of the cornea changing process after the corneal endothelial cells (CECs) was destroyed by phacoemulsification surgery;It is to explore the method of the rhesus monkey vascular endothelial cell transplantation to substitute the allogeneic CECs,and the feasibility of anterior chamber injection transplantation method,through observe the position,morphologic and functional changes of the rhesus monkey vascular endothelial cell which was transplanted to the inner surface of cornea without CECs.Method:The RF\6A was cultivated to proliferation and marked by Brdu in vitro.The experimental monkeys are divided into3groups:experimental group (group A,3monkeys),control group(group B,2monkeys),blank control group(group C,4monkeys).In group A:the CECs was destroyed by phacoemulsification surgery;and the cultured RF\6A,marked by brdu,were transplanted onto the inside surface of cornea without CECs though anterior chamber injection transplantation method,then make the experimental animals facing down for3-4hours.In group B:one month after phacoemulsification surgery,using the methods the same as group A to transplant the cultured RF\6A which marked by Brdu.In group C:CECs was destroyed by phacoemulsification surgery but without cultured RMVECs transplantation.One of the group C rhesus monkeys was picked the experimental eye immediately for various testings afer the surgery.Corneal transparence of every monkey was frequently observed postoperation.Aqueous humor was collected in preoperation and1,2,3,4,8,12 postoperation weeks to measure the concentration of vascular endothelial growth factor(VEGF) in ELISA method. Six ions concentration were tested useing automatic biochemistry analyzer.Then all data proceed with data analysis.On4,8,12postoperative weeks,the experimental eyes were respectively detached to observe the distribution and morphological structure of the RF\6A in the inner surface of cornea.and morphological structure of both iris and trabecular meshwork by pathological section. anti Brdu monoclonal antibody immunohistochemistry,scanning electronic microscope (SEM) and transmission election microscopy(TEM).Result:Corneal transpareney:In the experimental observation period(3months), group A and group B had better transparency than group C, with normal anterior chamber depth and without corneal neovascularization and bullous kerat-opathy.Pathological section:Cell layer with Brdu staining positive was found on the posterior surface of cornea in group A and group B,indicated the cells are RF\6A.The Descemet's membrane was basically complete.Mild edema was occurred in the iris.There wasn't cellular debris blocking trabecular meshwork and the angle of anterior chamber structure was clear.Only residual CECs with Brdu staining negative were foud on the peripheral posterior surface of cornea in group C. The Descemet's membrane was gradually dissolved and disappeared except the cornea which was immdiately picked off in postoperation.Both iris and trabecular meshwork had clear structure.SEM:Group A and group B showed that RF\6A with irregular shape uniformly distributed on the inner surface of cornea and growing well.Scattered RF\6A grown on the iris surface but the structure of iris surface had not significant changes.There was not cellular debris blocking trabecular meshwork.Group C showed with only residual CECs which structure and morphology were distroyed. The Descemet's membrane was gradually disappeared without new CECs growing on the inner surface of cornea.TEM:The cultured RF\6A and which in posterior surface of cornea were irregular oblateness.Abundant organelles and characteristic WBPs appeared in cytoplasm suggested the characteristic and vitality of vascular endothelial cells in vivo.Only residual CECs which cytoplasmic edema and scarce organelles had been fouded in posterior surface of cornea in group C.The concentration of VEGF in aqueous humor show no significant difference among these three groups,but significant difference among different time points in the group A.Among these three groups Ca2+,Mg2+,P,Na+,K+,C1-concentration show no significant difference.but the Na+concentration in group A is slightly lower than in the group C on the3postoperation months.And the Mg2+,P concentration in group A have some changes in some extent.Conelusion:Anterior chamber injection transplantation method can transplant the rhesus monkey vascular endothelial cell into inner surface of cornea,and they can grow well on the Descemet's membrane which CECs were destroyed by phacoemulsification surgery.lt also can play a role of barrier to keep the corneal dehydration and transparency to a certain extent.There are not pathological influences to the morphological structure of iris and the anterior chamber angle in the short time after the transplantion surgery.But it shows some influence to the concentration of VEGF,Ca2+,Mg2+,P,Na+,K+,C1-in aqueous humor.