| Objective: To investigate the influence of water soluble azone on the corneal endothelium cells;To evaluate the efficiency of liposome mediated gene transfer to corneal endothelial;To study azone promoting plasmid DNA transfection mediated with liposomes,and investigate the effect of ionizing radiation on liposome-mediated gene delivery and find out a way to improve gene transfection.Methods: Rabbit endothelium cells were cultured for in vitro test. Different concentration of azone was added to the culture medium to test its influence on the cell vitality and ultramicro structure, in which MTT methods, scanning and transmission electron microscope were used. With green fluorescence protein(GFP) as report gene,the transfection condition was optimized in cell confluence,liposome quantity,liposome/DNA ratio and transfecting time; Dispensing varied transfection agents of liposome+ DNA plasmid pEGFP-N1 , azone + liposomes + pEGFP-N1 , azone+ pEGFP-N1 , pEGFP-N1 , liposome merely and azone sole..Rabbit corneal endothelial cell were treated with the agents respectively. Green fluorescence protein (GFP) gene as a report gene was detected by fluorescence microscopy and flow cytometry, The transfection efficiency was figured out and compared with each other.Results: After incubated with water soluble azone under 0.1g.L-1 for 24 hours , the cell vitality of rabbit corneal endothelium was not significantly affected, while cell vitality was significantly suppressed with azone of 0.5g.L-1 and higher concentration(P<0.01). Corneal endothelium cells incubated with water soluble azone of no higher than 0.1g.L-1 concentration for 10 minutes, little slits and holes were detected on the plasma membrane by scanning electron microscope but no toxic effects was detected of the cell organelle (mitochondrion et al) with transmission electron microscope . While the concentrations of water soluble azone were 0.5g.L-1 and higher, most of the cells died. The GFP expression in corneal endothelial cells was observed, The highest transfecting efficiency of LipofectAMINE2000 would be obtained when cell confluence was80%～90% , liposome concentration1.8μl, ratio of liposome/DNA3μl/μg and transfecting time was 5 hours. Prior to liposome transfection, CECS were incubated with azone for 5 min; GFP was not detected in corneal endothelial cell treated with agents of merely liposomes and azone. Transfection rates of GFP in corneal endothelial cells treated with agent of azone + liposomes + pEGFP-N1 were 42.6% . With liposome + pEGFP-N1 , the rates of transfection in corneal endothelial cells were 21.4 % ,. The expression of GFP was7.2 % in corneal endothelial cells treated with azone + pEGFP-N1. With agent of azone + liposomes + pEGFP-N1 , a high transfetion rate of GFP gene was obtained. And no significant influence on the vitality of corneal endothelium cells was observed.Conclusion:Water soluable azone at concentration of no higher than 0.1g.L-1 has no significant influence on the vitality and ultrastructure of corneal endothelium cells . Azone could make slits and holes on the cell membrane without toxic effects on corneal endothelial cells. Azone may be used to help the gene transfer agent for the gene therapy research of corneal endothelium cells and for other use. Liposome is able to deliver genes to corneal endothelial cells with efficacy, the experiment implied result show that GFP a convenient reporter gene. Azone may enhance transfection rate of plasmid DNA mediated with liposome, azone has no inhibitory effect to CECs in vitro with efficient concentration and may be a new agent to gene transfection.
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