The Feasibility Study Of The Rat Bone Marrow Mesenchymal Stem Cells Directional Differentiate Into Corneal Endothelial Cell In Vitro

Objective:The purpose of this study was to investigate whether rat bone marrowmesenchymal stem cells (rBMSCs) can be directionally differentiated into cornealendothelial cell (CECs) in vitro, Mastering cell growth characteristics,detecting neuroncharacteristics enolase (NSE), protein expression of Na+-K+-ATPase enzyme activity,expression of the CD31and CD34convection antibody and to study the possiblemechanism.Methods:1.rBMSCs was separated and isolated by Percoll separation liquid from rat bonemarrow of SD rats from Dalian medical university Laboratory, then cultured andamplificated in vitro. The cell morphology was observed with inverted microscope andBMSCs were identified by detecting CD29、CD105、CD44、CD34antibody with theflow cytometry instrument.2.rBMSCs were planted in12-well plate by density of2×105/ml,then dividedinto induced group and the control group. It was covered about80%of the bottom ofthe bottle after3days and inducer added to Medium that was containing10ng/mlEGF,30ng/ml bFGF was induced differentiation culture, then2days change the firstliquid and theI nduced cycle was3weeks.3.Three weeks later, the differentiated cells were detected by immunofluorescenceand immunohistochemistry to test the expression of Na+-K+-ATPase and NSErespectively, to test the expression of endothelial cell markers CD31,CD34by means offlow cytometry instrument.Results:1.The separated rBMSCs were arranged in a spiral shape.The central cells were triangular and short spindle.CD29、CD105、CD44surface antigen expression rateswhich were tested by flow cytometry instrument were85.4%,91.2%,90.1%respectively.CD34surface antigen expression rate was1.2%, which were showed thatrBMSCs were successfully isolated and cultured.2.After2weeks,the induced rBMSCs detected under microscope showed somecells induced into polygon and had the characteristics of endothelial cellmorphology.After3weeks, the induced rBMSCs was "paving stones" shape,and themajority were hexagon.At the same time, immunofluorescence staining experimentsshowed that CD31and the expression of NSE positive rates were (10.48±0.19）%,（16.71±1.85）%respectively, while in control group CD31and NSE antibody positivecolor rates were negative.There were significant differences between induced groupand control group(X2=23.17,p<0.05).Na+-K+-ATPase activity expressed in inducedgroup was(0.231±0.107)U/mgprot, while in control group was0U/mgprot and there wassignificant difference between induced group and control group (t=41.67, p<0.01).CD31and CD34surface antigen detection by means of flow cytometry instrumentpositive expression rates in induced group were85.6%and92.4%. In blank group were1.2%and1.4%.Conclusion:The rat bone marrow mesenchymal stem cells(rBMSCs) can be successfullyseparated in vitro.rBMSCs have the potential to differentiate into corneal endothelialcells.The expression of the NSE，Na+-K+-ATPase activity,CD31and CD34convectionantibody is positive by induced bone marrow mesenchymal stem cells.Induceddifferentiation of rBMSCs can be used as seed cells,which can be possible to transplantcorneal endothelial cells in clinic.