Expression Of Enhanced Green Fluorescent Protein Gene In Guinea Pig Fibroblasts Mediated By Adeno-associated Virus

Objective: Comparison of the transfection efficiency and safety of enhanced green fluorescent protein(EGFP)gene in guinea pig scleral fibroblasts(GSF)mediated by four different adeno-associated virus(AAV).Methods: GSF were cultured in vitro from 2 weeks old normal tricolor guinea pigs,2generations were used for the experiments.The cells were exposed to different kinds of AAV mediated EGFP in transfection groups and the same quality of blank culture medium in control groups.At different multiplicity of infection(MOI:10,100,1000,10000)and various time points(2 days,3 days,4 days and 5 days)after infection,we observed the expression of GFP by inverted fluorescence microscope.Transfection efficiency and apoptosis rate were determined by flow cytometer.Cell viability was determined by CCK8.Results:(1)Observation by fluorescence microscope.As four kinds of AAV-EGFP transfected cells for 24 hours,the green fluorescence could be seen.The transfection efficiency increased with time and MOI,It went to peak at the 5th day in AAV2-EGFP group,AAV5-EGFP group and AAV8-EGFP group.AAV8-EGF group showed a significantly higher fluorescence intensity compared to the AAV2-EGFP group and AAV5-EGFP group.A weak fluorescence was observed in the AAV9-EGFP group.No fluorescence was observed in the control group.(2)Detection of transfection efficiency by flow cytometry.At 5th day(MOI=10000),the transfection rates were as follows: AAV2-EGFP group(31.433 ± 2.742)%,AAV5-EGFP group(21.633 ± 0.907)%,AAV8-EGFP group(74.867 ± 4.601)%,AAV9-EGFP group(4.650 ± 0.205)%.There is significant difference between groups.AAV8-EGFP group was higher than the other three groups,the difference is statistically significant,P <0.05.(3)Detection of apoptosis rate by flow cytometry.At 5th day(MOI=10000),the apoptosis rates were as follows : AAV2-EGFP group(4.990 ± 1.432)%,AAV5-EGFPgroup(5.960±0.567)%,AAV8-EGFP group(5.490±1.233)%,AAV9-EGFP group(5.200±1.186)%,control group(4.60±0.74)%.There is no significant difference in apoptosis rate between groups,P> 0.05.(4)Detection of cell viability by CCK8.At 5th day(MOI= 0,10,100,1000,10000),the OD values measured at 450 mM of AAV2-EGFP group: 0.478±0.019,0.466±0.023,0.459±0.041,0.465±0.029,0.471±0.021;AAV5-EGFP group: 0.475±0.006,0.500±0.018,0.477±0.022,0.453±0.044,0.474±0.007;AAV8-EGFP group: 0.479±0.014,0.476±0.026,0.476±0.017,0.474±0.019,0.480±0.039;AAV9-EGFP group: 0.481±0.003,0.488±0.010,0.442±0.047,0.479±0.025,0.480±0.052.There is no significant difference in apoptosis rate between groups,P> 0.05.Conclusion:(1)The ability of four adeno-associated viruses to mediate EGFP gene transfection into GSF: AAV8>AAV2>AAV5>AAV9.(2)AAV2,AAV5,AAV8 and AAV9 have no influence on cell viability and apoptosis rate.