Effects of conbercept combined with PDGF receptor inhibitor on the proliferation, migration of RPE cells and VEGF expression in hypoxic conditions / 眼科新进展

ABSTRACT

Objective To investigate the effects of conbercept combined with platelet-derived growth factor (PDGF) receptor inhibitor on the proliferation and migration of human retinal pigment epithelial cells (ARPE-19),as well as mRNA and protein expression of vascular endothelial growth factor (VEGF).Methods ARPE-19 cells were cultured in in vitro and the cells in logarithmic growth phase were obtained and induced by different concentrations of CoCl2.Then cell proliferation and toxicity test kit (CCK-8) was used to detect the proliferation activity of ARPE-19 cells for screening the optimal concentration of CoCl2 to construct hypoxic model.The cultured ARPE-19 cells were divided into normal group,hypoxic group and hypoxia + conbercept group (different concentrations),and CCK-8 assay was applied to analyze the effects of conbercept with different concentrations on cell proliferation activity for screening the best concentration of conbercept.The ARPE-19 cells in logarithmic phase were divided into normal group,hypoxic group,hypoxia + PDGF receptor inhibitor group (different concentrations),and CCK-8 assay was performed to detect the effects of PDGF receptor inhibitor on cell proliferation with hypoxic damage activity for screening the best concentration of PDGF receptor inhibitor.The logarithmic growth phase cells were divided into normal group,hypoxic group,hypoxia + 20 mg · L-1 conbercept group,hypoxia + 200 μmol · L-1 PDGF receptor inhibitor group,hypoxia + conbercept + PDGF receptor inhibitor group (both optimal concentration),and then the cell proliferation in each group was detected by CCK-8 assay,and the migration was detected by transwell chambers.The level of VEGF protein in the supernatant of each group was detected by ELISA methods,and VEGF mRNA expression was measured by RT-PCR.Results CCK-8 assay results showed that the A value of ARPE-19 in the 100 μmol · L-1 CoCl2 group was the highest (1.063 ± 0.031),which was set to be the optimal concentration for preparation of hypoxic model.CCK-8 assay results showed the cell proliferation rate of 20 μg · mL-1 conbercept and 20 μmol · L-1 PDGF receptor inhibitor group was (94.58 ± 3.80) ％ and (96.72 ± 5.44) ％,respectively,which could achieve the most satisfying efficacy,thereby both concentrations were selected for follow-up experiments.The cell proliferation and migration ability and VEGF protein level decreased in the conbercept group,PDGF receptor inhibitor group and conbercept + PDGF receptor inhibitor group when compared with the hypoxic group,and the decrease in the combined group was the most significant.Moreover,VEGF mRNA expression in the conbercept group and combined group were decreased when compared with the hypoxic group.Conclusion Hypoxia can enhance cell proliferation and migration ability and induce the up-regulation of VEGC protein and mRNA expression.In addition,PDGF receptor inhibitor combined with conbercept has inhibitory effects on the migration and proliferation of ARPE-19 cells as well as VEGF protein and mRNA expression following hypoxia injury,which is superior to conbercept treatment alone.