Study On The Bone Marrow Mesenchymal Stem Cell Of Rhesus Monkey Induce To Corneal Endothelial Cell In Vito And Vitro

Purpose:To examine the feasibility of the corneal endothelial cells substituted with bone marrow mesenchymal stem cells(BMSCs) with the method of transplanting rhesus monkey BMSCs culturaled in vitro into the endothelial surface of corneal whose Descemet membrane was splited,through observing the change of cell form and function in different time. And the same time, to explorate the environmental conditions that BMSCs induced to corneal endothelial cells(CECs) in vitro preliminarily.Methods:The experimented monkeys are divided into2groups:experimental group (6monkeys) and control group (2monkey). Bone mesenchymal stem cells (BMSCs) were isolated from bone marrow by density gradient centrifugation combined with adhering means. After identification and labeled with5-bromodeoxyuridine (Brdu),the cultured cells were transplanted onto the endothelial surface of corneal whose CECs has been removed. Post-operation, the transparency of corneas were observed, corneal graft were remove after1month,2month,3month respectively for measurement purpose. Such as pathology detection, anti Brdu monoclonal antibody staining, electron microscopy observation of the distribution and the connection between transplanted cells and the changes of their morphology. There are two trial method in vitro to induce the differentiation,the first is putting the CECs together with BMSCs into Transwell co-cultral system,the other is transferring the BMSCs into a special medium in which aqueous fluid achieved from the anterior chamber of decompensated cornea were incorporated. Detect the BMSCs with morphology and NSE to evaluate the examinated effect every session.Result:Corneal endothelial function decompensation model was made after corneal endothelial being damaged by phacoemulsification, and the corneal transparency gradually declines. After BMSCs transplanted in anterior chamber, the experiment group corneal transparency was gradually increased, which proved that the transplanted cells were well-grown at the damaged corneal surface and play a role. The follows would be seen when we observed using electron microscope:The growth of cells stacked in the first month, and there were few connections between cells. The connections between cells increased compared with the previous in the second month. The cells grew like monolayer, and there were more connections between cells, but cells window exists. On the form, it is Gradually from long to short spindle cells and polygon. Cornea of the matched group was turbid and couldn’t recover, meanwhile new blood vessels ingrowth could be seen. Descemet’s membrane was bare when observed under electron microscope, no cells covered, and residual corneal endothelial cells may be seen occasionally with weak attached. Two methods of in vitro induction got positive results dedected with NSE in2weeks and3weeks, and the cell morphology change was not obvious.The repeat induced experiment result was the same with the above.Conclusion:Allogeneic BMSCs cultured in vitro can partly replace corneal endothelial cells transplant into corneal internal surface with anterior chamber injection method,moreover play the pump function and express part of the related proteins of surface endothelial cell. Both Transwell indirect co-culture and Aqueous humor-medium can induce BMSCs into corneal endothelial cells in vitro.