Preliminary Study On The Expression Of VEGF164and SEMA3A Induced By CoCl₂in Rat Retinal Progenitor Cells

AIM: To investigate the effect of hypoxic environment simulated by CoCl₂on the expression ofthe vascular endothelial growth factor （VEGF） and axon guidance factor （SEMA3A） of ratretinal progenitor cells（RPCs）, and to explore the relationship and interactions between VEGFand SEMA3A under hypoxic conditions and its significance.METHODS: RPCs were separated, cultured and identified by the use of neurospheres-adhesiveculture. To identify RPCs by the means of Immunohistochemistry and to detect the expression ofVEGF164, SEMA3A and their common receptor NRP-1. CCK8was used to detect the change ofRPCs’ viability after24-hour intervention of CoCl₂at different concentrations （0,50,100,150μmol/L and200μmol/L） or the intervention of150μmol/L CoCl₂for different periods of time（0、12、24、36、48、72h）. RealTime-PCR and ELISA was applied to detect the mRNA and proteinexpression of VEGF164, SEMA3A in RPCs of each group.RESULTS: Strong expression of neural stem cell specific marker （Nestin） was discovered inRPCs cultured by neurospheres-adhesive culture system, RPCs could also strongly expressVEGF, SEMA3A and NRP-1, as well as retinal cell antigen Rhodopsine, MAP-2and GFAP afternatural differentiation. When the CoCl₂was at certain concentrations （0²00μ mol/L） andcultured for certain time （0⁴8h）, the survival rate of RPCs was not affected; After culturing inthe hypoxic environment for the same period, the expression of VEGF mRNA graduallyincreased and then decreased with the increasing concentration of CoCl₂, the expression ofSEMA3A decreased and then increased with the increasing concentration of CoCl₂. When theconcentration of CoCl₂was150μmol/L, the expression of VEGF mRNA also graduallyincreased and then decreased with the increasing time of intervention, the expression ofSEMA3A decreased and then increased with the increasing time of intervention. The changes ofthe expression of protein and gene tend to be consistent.CONCLUSION: The RPCs cultured with the neurospheres-adhesive culture method can betterexpress the specificity of stem cells and the normal RPCs cultured in vitro express VEGF164, SEMA3A and NRP-1. The detrimental effect of CoCl₂treatment of certain concentrations anddurations on the viability of RPCs was not obvious. Under the hypoxic environment simulatedby CoCl₂, the secretion of VEGF164and SEMA3A of RPCs was dependent on the time and dose.The expression of VEGF164may be reduced by the up-regulation of SEMA3A, which may beone of the protective mechanisms against neovascular retinopathy.