The Effects Of Slit2 Gene In RPE Cells On The Migration And Tube Formation Of RF/6A Cells

Objective:To observe the effects of slit2 gene in retinal pigment epithelial (RPE) cells on migration and tube formation of choroidal vasular endothedial cells (RF/6A) in a coculture system.Methods:Transfect ad-slit2 or ad-null to RPE cells for 24、48h, the mRNA and protein of slit2 were quantified by RT-PCR and Western blot. Two kinds of coculture models (RPE&RF/6A) were used to observe the effects of RPE cells transfected with ad-slit2 on the migration and tube formation of RF/6A cells respectively. Then establish chemical hypoxia model in vitro, transfected shRNA-slit2 or GFP to the model, RT-PCR and Western-blot detected the mRNA and protein of slit2 and VEGF, and the level of secreted VEGF protein from the media were investigated by ELISA. In the coculture system (RPE&RF/6A), observe the effects of RPE cells transfected with shRNA-slit2 or GFP on the migration and tube formation of RF/6A cells.Results:After ad-slit2 transfected to RPE cells for 24h and 48h, it resulted the increasing of the corresponding mRNA and protein of slit2. In the coculture system, the number of RF/6A cells migrated in the blank control group, ad-null for 24h group, ad-slit2 for 24h group, ad-null for 48h and ad-slit2 for 48h group was respectively (40.6±4.5),(36.7±3.5),(67.7±4.2), (36.0±3.6), (79.7±3.5), and the tube formation number of RF/6A cells was respectively (8.7±3.2)、(8.9±3.6)、(15.9±4.2)、(8.3±2.4)、 (23.3±4.5). There was significant difference between ad-slit2 for 24h group,ad-slit2 for 48h group and blank control group statistically (P<0.05). Under hypoxia, the mRNA and protein of slit2 decreased after transfection with shRNA-slit2. In the coculture model, the number of RF/6A cells migrated in the blank control group, hypoxia group, GFP under hypoxia group, shRNA-slit2 under hypoxia group was respectively (36.7±5.5)、 (110.0±8.0)、(104.6±14.6)、(61.0±5.3); and the tube formation number of RF/6A cells was respectively (6.7±1.5)、(19.7±3.5)、(21.3 ±3.8)、(10.3±1.6). There was significant difference between shRNA-slit2 under hypoxia group and hypoxia group statistically (P<0.05). After shRNA-slit2 transfected to RPE cells under hypoxia, it resulted the decreasing of the mRNA and protein of VEGF. At the same time, the level of secreted VEGF protein from the media of RPE cells also declined.Conclusion:slit2 gene in RPE cells can promote the migration and tube formation of RF/6A cells in vitro. Under hypoxia, down-expresssion of Slit2 gene in the RPE cells can inhibit the previous mentioned progress of RF/6A. Slit2 gene in RPE cells may participate in the formation of CNV.