Role Of Shh Signal Transduction Pathway In VEGF Expression Under Hypoxia In Cultured Human Retinal Pigment Epithelial Cells

Objective To investigate the role of Sonic Hedgehog(Shh)signal transduction pathway in VEGF expression under hypoxia in cultured human retinal pigment epithelial(hRPE)cells.Methods ARPE-19 were cultured and divided into normal ARPE-19(Cont)and hypoxia group(100umol·L-1 CoCl2 Cobalt Chloride +ARPE-19);Hypoxia group divided into CoCl2 group,Cyclopamine group(CYA)and DMSO group.Only CYA group added 20?mol/L Cyclopamine 1 hour before hypoxia,added 1‰DMSO into DMSO group at the same time.The hRPE cells were cultured under hypoxia for 0 hour?4 hour?8 hour?12 hour and 24 houn.The expression of Shh and VEGF were determined by Real-time fluorescent quantitate polymerase chain reaction.The amount of VEGF in the hRPE-conditioned supernatant was measured using enzyme linked immunosorbent assay at 4 hour?8hour?12 hour and 24 hour,respectively.Results RT-PCR tests showed:Under hypoxia,the level of SHH and VEGF of hRPE was time dependently increased(Shh:F=45.260 P=0.001;VEGF:F=264.938 P=0.001).The level of Shh and VEGF of hRPE in the group treated with Cyclopamine was decreased(4?8?12?24h Shh mRN A:P=0.018;P=0.001;P=0.001;P=0.001 VEGF mRNA:P=0.001;P=0.001 P=0.001;P=0.001).ELISA tests showed:the amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner(F=3156.676 P=0.001),and it was down-regulated by Cyclopamine under hypoxia(4?8?12?24h VEGF Protein:P<0.01)· Conclusions Sonic Hedgehog(Shh)signal transduction pathway could play a role in the VEGF expression induced by hypoxia in hRPE cells.