Co-transfection Of Humanβ-NGF And PDGF-B Gene In Cat Corneal Endothelial Cells Via Adeno-associated Virus Mediation

PURPOSE:To research the biological effect of co-transfection of adeno-associated virus (AAV) mediated human nerve growth fector-B (β-NGF) and human platelet derived growth factor B (PDGF-B) in cat corneal endothelial cells in vitro.METHODS:Cat corneal endothelial cells were cultured in vitro. The type and purity of the endothelial cells were further confirmed by morphological analysis and NSE immunofluorescence study. After forming the adeno-associated virus vector, cat corneal endothelial cells were transfected by AAV mediated green fluorescent protein (GFP) gene. Then the efficiency of transfection was evaluated according to positive expression of GFP. Humanβ-NGF gene and human PDGF-B gene were transfected into cat corneal endothelial cells in vitro by AAV vector. Real-Time PCR and Western blot were used to check the expression status ofβ-NGF and PDGF-B on mRNA and protein level in cat corneal endothelial cells at 24 hours,48 hours and 72 hours after respective transfection and co-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 72 hours after respective transfection and co-transfection of AAV-β-NGF and AAV-PDGF-B. By cell scratch test, the effect of cell spreading ability in cat corneal endothelial cells after transfection was detected.RESULTS:Cat corneal endothelial cells could be rapidly cultivated to form pure single layer. The type and purity of cells were further confirmed by morphological analysis and NSE immunofluorescence study. Cat corneal endothelial cells displayed green fluorescence clearly under fluorescence microscope in 72 hours after transfection of AAV mediated GFP gene. The efficiency of transfection reached 67.8%. The results of Real-Time PCR and Western blot showed that the expression ofβ-NGF and PDGF-B increased with time extended. Compared with the control groups, the difference had statistical significance (P< 0.05). There were no significant difference (P>0.05) between the expression ofβ-NGF and PDGF-B in co-transfection groups and respective transfection groups at 24 hours,48 hours,72 hours. The results of MTT showed that the proliferation ability of cells increased after the transfection of AAV-β-NGF and AAV-PDGF-B, moreover co-transfection groups increased obviously. There were statistical difference(P<0.05)between the each transfection groups and the control groups. The results of flow cytometry showed that the cell number at Gl stage increased after the transfection of AAV-B-NGF and AAV-PDGF-B, and the co-transfection groups increased more obviously than others. The results of cell scratch test showed the effect of cell spreading ability in cat comeal endothelial cells was increased after the transfection of AAV-β-NGF and AAV-PDGF-B, especially co-transfection groups. The cell spreading ability of each groups had statistical difference (P<0.05) between the transfection groups and the control groups.CONCLUSIONS:Humanβ-NGF gene and human PDGF-B gene could be effectively transfected into cat corneal endothelial cells in vitro by AAV vector, and expressed stably. Co-transfection of AAV-B-NGF and AAV-PDGF-B could increase the ability of proliferation and spreading in cat corneal endothelial cells.