Effect of hypoxia and high glucose on proliferation of retinal pigment epithelial cells and expression of VEGF and TGF-β1

Objective To investigate the cell growth and proliferation of retinal pigment epithelium(RPE)and the expression of vascular endothelial growth factor(VEGF)and transforming growth factor-β1(TGF-β1)under hypoxia and high glucose environment.Methods RPE cells of C57 mice were primary cultured in vitro with enzyme digestion.The cultured RPE cells were identified with histomorphological observation and immunocytochemical staining.In experimental hypoxia groups,the concentrations of CoCl2 added were 50 μmol·L-1,100 μmol·L-1,200 μmol·L-1 in DMEM culture fluid.In experimental high glucose groups,the concentrations of D-glucose added were 15 mmol·L-1,30 mmol·L-1,45 mmol·L-1 in DMEM culture fluid.Each of above-mentioned culture fluid was added to cultured RPE cells,and there was 6 groups,triplicate for each group.The culture fluid of normal control group contained 5.5 mmol·L-1 glucose,but without CoCl2.The vital force,doubling times and proliferation were observed.The expressive changes of VEGF,TGF-β1 in the supernanant at different time(24 hours,48 hours,72 hours)were also examined by ELISA.Results The primary and the first generation RPE cells grew well;Cells cultured in hypoxia or high glucose medium reduced in vital force and population doubling,but lengthened in doubling time;Expression of VEGF and TGF-β1 in the hypoxia and high glucose groups were higher than those in the control group after 24 hours,48 hours,72 hours(P0.05);The peaks appeared in 100 μmol·L-1 CoCl2 group[(43.28±0.88)ng·L-1 and(8.90±1.38)ng·L-1 respectively after 48 hours] and 30 mmol·L-1 high glucose group[(39.76±0.56)ng·L-1 and(8.81±0.92)ng·L-1 respectively after 48 hours]at different time,comparing with other concentrations.The peak of cellular factors expression appeared at 48 hours in hypoxia group,and at 72 hours in high glucose group.Conclusions Purified RPE cells are obtained by enzyme digestion;Both hypoxia and high glucose environments can obviously inhibit the proliferation of the RPE cells,but increase the expressions of VEGF and TGF-β1.