The Ex Vivo Experiment About LENTI-EGFP Transfecting In Corneal Epithelial Cell

Objective To observe the effectivity and safety about lenti-EGFP transfection on corneal epithelial cell ex vivo.Methods We cultured primary and passage cell of rabbit corneal epithelial tissue, and the cells were transfected by lenti-EGFP in different Multiplicity of Infection (MOI=0, 1, 10, 50, 100, 500). We get the optimum multiplicity of infection and observe the expression of the enhanced green fluorescence protein (EGFP) through the Inverted fluorescence microscope, and calculate the infection rate in different MOI. At last, the target gene-EGFP was tested by the RT-PCR. The shapes and ultra microstructures of the epithelial cells were observed by HE stain and transmission electron microscope, and Flow cytometer tests the apoptosis of infected cell.Results The expression of EGFP was observed in the inverted fluorescence microscope after 48h of transfection. There is no distinct difference between MOI=100 and MOI=500(P>0.05), and the optimum MOI=100.When MOI=1, 10, 50,100, the transfection rate has the time-dependency and dose-d ependency (P< 0.05) . The outcome of RT-PCR clues to the expression of the EGFP gene. When MOI=100, the shape and ultra microstructures of infected cells are not changed significantly compare with the normal epithelial cells. The outcome of apoptosis rate test suggests that there are not significant difference in both normal and infected cells (P>0.05).Conclusion The lenti-EGFP may transfect the rabbit epithelial cell ex vivo efficiently, and the safety can be further approved.