Effectiveness and toxicity of the transfection of lentiviral vector-mediated enhanced green fluorescent protein to rat cornea / 解放军医学杂志

ABSTRACT

Objective To investigate the effectiveness and toxicity of the transfection of lentiviral vector-mediated enhanced green fluorescent protein (LV-EGFP) to rat cornea with different concentrations and methods, and to look for the most effective way for transfecting the virus to rat cornea. Methods Twenty-five SD rats were randomly divided into five groups, namely MOI=5 eyedrop group (group A), MOI=5 subconjunctival injection group (group B), MOI=10 eyedrop group (group C), MOI=10 subconjunctival injection group (group D), and MOI=10 in vitro transfection group (group E). Rats in each group were treated with corresponding concentration of LV-EGFP respectively. The distribution and intensity of fluorescence in the corneas were observed under inverted fluorescence microscope, and the corneal cellular morphology was observed with HE staining 7 days after transfection. Results Within the same MOI groups, fluorescence spread more uniformly in eyedrop groups as compared with that in subconjunctival injection groups. The fluorescence intensity (A value) in five groups were 0.1803±0.0440, 0.1061±0.0434, 0.2369±0.0157, 0.2002±0.0307, 0.2434±0.0173 respectively, which was stronger in eyedrop groups than in subconjunctival injection groups (P<0.05). In high MOI groups more EGFP could be expressed as compared with low MOI groups (P<0.05). The fluorescence intensity was significantly higher in group E than in groups A, B and D, while there was no statistical significant difference between groups E and C. HE staining showed no apoptotic corneal endothelial cells. The corneal endothelial cells were still growing well with no shrinkage, deformation and loss in vitro 7 days after transfection. Conclusion Rat cornea could be effectively transfected by LV-EGFP with low MOI. Eyedrop method is found to have a higher efficiency compared with subconjunctival injection. An increase in amount of MOI can increase transfection efficiency. Continuous transfection with low MOI to cornea of rat is safe.