康柏西普联合PDGF受体抑制剂对缺氧条件下视网膜色素上皮细胞增殖、迁移及VEGF表达的影响

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

目的探讨康柏西普联合血小板衍生生长因子(platelet derived growth factor,PDGF)受体抑制剂对CoCl_2诱导的人视网膜色素上皮细胞ARPE-19细胞增殖、迁移以及血管内皮生长因子(vascular endothelial growth factor,VEGF)m RNA及蛋白表达的影响。方法体外培养ARPE-19细胞,取对数生长良好的细胞,用不同浓度CoCl_2诱导培养,细胞增殖与CCK-8法检测ARPE-19细胞增殖活性,筛选最佳CoC l2浓度用于构建缺氧模型;将培养的ARPE-19细胞分为正常组、缺氧组、不同浓度康柏西普组,CCK-8法检测不同浓度康柏西普对缺氧损伤后ARPE-19细胞增殖的影响,筛选最佳康柏西普浓度;将培养的ARPE-19细胞分为正常组、缺氧组、不同浓度PDGF受体抑制剂组,CCK-8法检测不同浓度PDGF受体抑制剂对缺氧损伤后ARPE-19细胞增殖的影响,筛选最佳PDGF受体抑制剂浓度;将培养的ARPE-19细胞分为正常组、缺氧组、20 mg·L^(-1)康柏西普组、20μmol·L^(-1)PDGF受体抑制剂组、康柏西普联合PDGF受体抑制剂组(最佳药物浓度组合);CCK-8法检测缺氧损伤后各组ARPE-19细胞增殖活性,Transwell小室检测细胞的迁移情况,ELISA法检测细胞上清液中VEGF蛋白浓度,应用实时荧光定量PCR法检测细胞VEGF mR NA的表达水平。结果 CCK-8筛选结果显示,100μmol·L^(-1)CoC l2处理组ARPE-19细胞的吸光度值最大(1.063±0.031),设立为缺氧细胞模型浓度。CCK-8筛选结果显示,20 mg·L^(-1)康柏西普组细胞增殖率为(94.58±3.80)%、20μmol·L^(-1)PDGF受体抑制剂组细胞增殖率为(96.72±5.44)%,效果均较其他相应组好,选取该浓度进行后续实验。析因分析发现缺氧损伤后各药物处理组(康柏西普、PDGF受体抑制剂、康柏西普联合PDGF受体抑制剂)与缺氧组比较,细胞增殖活性下降,细胞迁移数降低,VEGF蛋白浓度亦下降,其中康柏西普联合PDGF受体抑制剂组下降最明显,康柏西普组及两药联合处理组ARPE-19细胞VEGF mR NA表达较缺氧组降低。结论缺氧损伤后可诱导ARPE-19细胞增殖、迁移,VEGF蛋白相对表达量增加,VEGF mR NA表达上调;康柏西普联合PDGF受体抑制剂对缺氧损伤后ARPE-19细胞的增殖、迁移及VEGF蛋白、VEGF mR NA的表达有抑制作用,作用效果优于单独应用康柏西普。

ObjectiveTo 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(ARPE19),as well as mRNA and protein expression of vascular endothelial growth factor(VEGF).MethodsARPE19cells 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(CCK8)was used to detect the proliferation activity of ARPE19cells for screening the optimal concentration of CoCl2to construct hypoxic model.The cultured ARPE19cells were divided into normal group,hypoxic group and hypoxia+conbercept group(different concentrations),and CCK8assay was applied to analyze the effects of conbercept with different concentrations on cell proliferation activity for screening the best concentration of conbercept.The ARPE19cells in logarithmic phase were divided into normal group,hypoxic group,hypoxia+PDGF receptor inhibitor group(different concentrations),and CCK8assay 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+20mg·L-1conbercept group,hypoxia+200μmol·L-1PDGF receptor inhibitor group,hypoxia+conbercept+PDGF receptor inhibitor group(both optimal concentration),and then the cell proliferation in each group was detected by CCK8assay,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.ResultsCCK8assay results showed that the A value of ARPE19in the100μmol·L-1CoCl2group was the highest(1.063±0.031),which was set to be the optimal concentration for preparation of hypoxic model.CCK8assay results showed the cell proliferation rate of20μg·mL-1conbercept and20μmol·L-1PDGF 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.ConclusionHypoxia 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 ARPE19cells as well as VEGF protein and mRNA expression following hypoxia injury,which is superior to conbercept treatment alone.