摘要
Purpose:To investigate the effect of small interfering RNA (siRNA)targeting VEGF of retinal pigment epithelium (RPE) cells on the growth activity of human retinal vascular endothelial cells (RECs) under a co-culture system.Methods:By applying the vector.(pGPU6)-based siRNA plasmid gene silence system,we specifically silenced VEGF expression of RPE cells (ARPE-19) through plasmid (pG-PU6-VEGFA-siRNA)transfection.Reverse transcription polymerase chain reaction(RT-PCR) was applied for selecting the most efficient siRNA segment among three pGPU6-VEGF-siRNA groups (siRNA-1,siRNA-2 and siRNA-3).Treated RPE cells were co-cultured with RECs in a co-culture system made up of a 24-well culture plate and transwell inserts assembled inside During 7-day culture period,the growth ca-pacity of RECs were observed and tested in the form of cell counting assay.Three groups were established in this study:RPE cells transfected with pGPU6-VEGF-siRNA and co-cultured with RECs (group A),RPE cells transfected with siR-NA null vector and co-cultured with RECs (group B),and RECs cultured alone (group C).Results:After transfection,VEGF expression levels of RPE cells in three pGPU6-VEGF-siRNA groups (siRNA-1,siR-NA-2 and siRNA-3)evaluated by RT-PCR were 2.56 ±0.45,1.17 ±0.38 and 4.39 ±0.51,respectively (n =10).siRNA-2 was selected as the foremost segment for transfection (P < 0.05,SNK-q test).During the 7-day co-culture period,the influence upon the growth of RECs was observed.Growth curve of RECs under co-culture showed a lower growth rate in group A than in group B (P <0.05,dunnett's test),but no significant difference between group A and group C was noted (P>0.05,dunnett's test).RECs in group A proliferated muchfaster during the first four days post-transfection.Conclusion:Delivery of siRNA targeting VEGF plays an efficient role in down-regulating VEGF expression in RPE cells,therefore modulating the growth activity of RECs under a co-culture system in vitro.The application of this technique may provide novel evidence for the prevention and treatment of retinal neovascularization diseases.
Purpose： To investigate the effect of small interfering RNA （siRNA）targeting VEGF of retinal pigment epithelium （RPE） cells on the growth activity of human retinal vascular endothelial cells （RECs） under a co-culture system. Methods： By applying the vector （pGPU6）-based siRNA plasmid gene silence system, we specifically silenced VEGF expression of RPE cells （ARPE-19） through plasmid （pG- PU6-VEGFA-siRNA）transfection. Reverse transcription polymerase chain reaction（RT-PCR） was applied for selecting the most efficient siRNA segment among three pGPU6-VEGF- siRNA groups （siRNA-1, siRNA-2 and siRNA-3）. Treated Rite cells were co-cultured with RECs in a co-culture system made up of a 24-well culture plate and transwell inserts assembled inside During 7-day culture period, the growth capacity of RECs were observed and tested in the form of cell counting assay. Three groups were established in this study： RPE cells transfected with pGPU6-VEGF-siRNA and co-cultured with RECs （group A）, RPE cells transfected with siR- NA null vector and co-cultured with RECs （group B ）, and RECs cultured alone （group C）. Results ： After transfection, VEGF expression levels of RPE cells in three pGPU6-VEGF-siRNA groups （siRNA-1, siR- NA-2 and siRNA-3）evaluated by RT-PCR were 2.56±0.45, 1.17±0.38 and 4.39±0.51, respectively （n=10）. siRNA-2 was selected as the foremost segment for transfection （P 〈 0.05,SNK-q test）. During the 7-day co-culture period, the influence upon the growth of RECs was observed. Growth curve of RECs under co-culture showed a lower growth rate in group A than in group B （P〈0.05,dunnett＇s test）, but no significant difference between group A and group C was noted （P〉0.05, dunnett＇s test）. RECs in group A proliferated much faster during the first four days post-transfection. Conclusion： Delivery of siRNA targeting VEGF plays an efficient role in down-regulating VEGF expression in RPE cells, therefore modulating the growth activity of RECs under a coculture system in vitro. The application of this technique may provide novel evidence for the prevention and treatment of retinal neovascularization diseases.
