title: The effect of siRNA-VEGF on the growth of REC in retinal pigment epithelial cell and retinal endothelial cell co-culture system.
abstract: OBJECTIVE
  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 (pGPU6-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 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 siRNA null vector and co-cultured with RECs (group B), and RECs cultured alone (group C).
  
  
  RESULTS
  After transfection, VEGF expression of RPE cells in three pGPU6-VEGF-siRNA groups (siRNA-1, siRNA-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, an 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.
  
  
  CONCLUSIONS
  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 neovascularisation diseases.
authors.affiliation: Eye Center, the Second Affiliated Hospital, School of Medicine of Zhejiang University, Hangzhou 310009, China. zj2majin@yahoo.com.cn
authors.forename: Jin
authors.initials: J
authors.lastname: Ma
authors.forename: Zhaomeng
authors.initials: Z
authors.lastname: Shen
authors.forename: Qianru
authors.initials: Q
authors.lastname: Zhang
authors.forename: Tiepei
authors.initials: T
authors.lastname: Zhu
authors.forename: Ke
authors.initials: K
authors.lastname: Yao
mesh_terms: D018920:Coculture Techniques; D015536:Down-Regulation; D042783:Endothelial Cells / Q000378:metabolism*; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D009389:Neovascularization, Pathologic; D010957:Plasmids; D034741:RNA, Small Interfering / Q000235:genetics*; D015861:Retinal Neovascularization / Q000517:prevention & control; D055213:Retinal Pigment Epithelium; D012168:Retinal Pigments; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D012168:Retinal Pigments; D042461:Vascular Endothelial Growth Factor A