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

Objective: To Explore the feasibility of rhesus monkey vascular endothelial cell(RMVECs) transplantation to substitute the allogeneic corneal endothelial cells(CECs), through observe the morphologic and functional change of the vascular endothelial cell which was transplanted to the inner surface of cornea without Descemet's Membrane.Method: The rhesus monkey vascular endothelial cell was cultivated to proliferation and marked by Brdu in vitro. The experimented monkeys are divided into 2 groups: experimental group (3 monkeys), control group (1 monkey). In experimental group: the cultured rhesus monkey vascular endothelial cell, marked by brdu, were transplanted onto the posterior surface of rhesus monkey cornea without Descemet's Membrane though centrifugal sedimentation, then sew back the corneal graft. In control group: detach the corneal Descemet's Membrane of rhesus monkey but without cultured RMVEC transplantation. Corneal transparence of every monkey was frequently observed postoperation. Aqueous humor was collected in preoperation, 7,14,21,30,45,60,90 postoperative days to measure the concentration of vascular endothelial growth factor (VEGF) in it useing ELISA. On 30,60,90 postoperative days, corneal graft were respectively detached to observe the distribution, appearance and ultrastructures morphological structure of RMVECs on the inner surface of cornea, by pathological section, anti brdu monoclonal antibody immunohistochemistry, scanning electronic microscope(SEM) and transmission election microscopy(TEM).Result: Corneal transparency: In the experimental observation period (three months), the corneal graft of experimental group had better transparency than control group and without corneal neovascularization and bullous keratopathy. Pathological section: A layer of cells with brdu staining positive was found on the posterior surface of cornea in experimental group, indicated the cells are RMVECs. And no cell-like structure was found in control group. Scanning electronic microscope: Experimental group showed that RMVECs with irregular shape uniformly distributed on the inner surface of cornea and growing well, a small amount of white blood cells can be seen between RMVECs, and part of cellular debris exist in the trabecular meshwork. Control group showed a fiber material without RMVECs. Transmission election microscopy: The cultured RMVECs and which in posterior surface of cornea was irregular oblateness, a large number of desmosomes link were been seen between RMVECs. Abundant organelles and characteristic WPBs appear in cytoplasm, which suggest the characteristics and vitality of vascular endothelial cells in vivo, and no cell structure in the control group. The concentration of VEGF in aqueous humor: The concentration of VEGF in aqueous humor between experimental group and control group show no significant difference on the first postoperation weeks, but significant difference on the 2,3,4,6,8,12 postoperation weeks, and the VEGF concentration of experimental group is significantly higher than control group in the corresponding time points.Conclusion: Rhesus monkey endothelial cells can growth on the posterior surface of cornea without Descemet's membrane. The cultured cells, with similar ultrastructure to RMVECs in vivo, can play a role of barrier to keep the corneal dehydration and transparency to some extent. The VEGF concentration in aqueous humor continues to rise within one postoperation month, and then stable at a high level. The treatment of vascular endothelial cell transplantation to replace the function of corneal endothelial cell, can provided a new way for treating corneal opacity clinically results from corneal endothelial diseases.