The Biological Effect Of Transfection With Adeno-associated Virus Encoding Human β-NGF On Cats Corneal Endothelial Cells

PURPOSE:To research the biological effect of transfection with adeno-associated virus (AAV) encoding human nerve growth factor-β(β-NGF) on cats corneal endothelial cells in vitro.METHODS:Cats corneal endothelial cells were cultured in vitro.The type and purity of the endothelial cells were further confirmed by morphological analysis,NSE immunohistochemistry study.Cats corneal endothelial cells were transfected by adeno-associated virus-mediated green fluorescent protein(GFP) gene,then the efficiency of transfection was evaluated according to positive expression of GFP.Humanβ-NGF gene was transfected into cats corneal endothelial cells in vitro by AAV vector.Real-Time PCR and ELISA were used to check the expression status ofβ-NGF at mRNA and protein level in cats corneal endothelial cells at 12 hours,1 day,3 days,5 days,7 days after transfection.MTT was used to detect the proliferation ability of cells,and flow cytometry was used to detect cell number at different stages of cell cycles at 3 days after transfection.RESULTS:Cats corneal endothelial cells could be rapidly cultivated to form pure single layer.The type and purity of cells were further confirmed by morphological analysis, NSE immunohistochemistry study.24 hours after transfection with AAV-mediated GFP gene,cats corneal endothelial cells displayed green fluorescence clearly under fluorescence microscope,the transfection efficiency can reach 67.5%.The results of Real-Time PCR and ELISA showed that the expression ofβ-NGF increased with time extended,and the maximum presented at the 5th day.Compared with control groups,the difference had statistical significance(P＜0.05);The results of MTT and flow cytometry showed that, after transfection,the proliferation ability of cells and the cell number at G1 stage increased obviously.Compared with control groups,the difference had statistical significance(P＜0.05).CONCLUSIONS:Humanβ-NGF gene could be effectively transfected into cats corneal endothelial cells in viro by AAV vector,and stably expressed to improve mitosis of the cat corneal endothelial cells.