A Role For Proliferation And Differentiation Experimental Study Of Notch Gene Transfected Rabbit Corneal Endothelia

This article aims to elect the strongest expression genes of the Notch family members, construct and transfect expression vector into rabbit corneal endothelia in vitro by lentiviral vectors transfection technique to identify that Changes in cell proliferation and differentiation, Explore the feasibility of transgenic technology in the treatment of corneal endothelial cell disease.Endothelial cells with Descemet,s membrane were mechanically Separated from the stroma cultured in vitro, Gene expression of Notch receptors and ligands was determined by PCR, gene expression vector was constructed and transfected into rabbit corneal endothelia in vitro by lentiviral vectors transfection technique. cell proliferation and differentiation were analyzed after RT-PCR,MTT and immunocytochemistry. Endothelial cells with Descemet,s membrane were mechanically Separated from the stroma could rapidly cultivate to form pure single layer rabbit corneal endothelia.Gene expression of receptors Notch2, Notch3 and ligands Delta1,Jagged1 was detected in cultured corneal endothelia cells, Notch3 performanced the strongest expression. after transfected 72 h,lentiviral vector transfection efficiency up to 60% at MOI = 100,.Cell differentiation gene α-SAM and Ki-67 expression up-regulation,whereas down—regulation cell junction marker gene ZO-1 and N-cadherin expression.lentiviral vector transfection technique could be effectively used in transfecting into rabbit corneal endothelia in vitro with good efficacy and stably expressed. Notch3 overexpression Improve the ability of Notch signal regulates corneal endothelia cells Proliferation and differentiation,which was blocked by the Notch inhibitor,DAPT. This results might provide a new theoretical basis of gene therapy for the corneal endothelial cell disease.