特异性bFGF拮抗剂FGFR1-Fc对视网膜新生血管的抑制作用

The inhibitory effect of specific bFGF antagonist FGFR1-Fc on retinal neovascularization

目的探讨特异性bFGF拮抗剂(FGFR1-Fc)对碱性成纤维细胞生长因子(bFGF)刺激的人视网膜微血管内皮细胞(HRCEC)增殖、迁移和血管生成的抑制作用。验证FGFR1-Fc在氧诱导的视网膜新生血管(OIR)小鼠模型中的抑制作用,并分析FGFR1-Fc和抗血管内皮生长因子(VEGF)药物的协同作用。方法体外培养HRCEC,用bFGF和不同浓度FGFR1-FC处理细胞。采用MTT法测定细胞活性、划痕法检测细胞迁移能力、流式细胞术检测细胞周期、蛋白质印迹法检测蛋白表达水平;以OIR小鼠为动物模型,观察视网膜血管状态及计算视网膜无灌注区面积。采用苏木精-伊红染色(HE)法计数突破视网膜内界膜的新生血管内皮细胞核数目。结果FGFR1-Fc抑制bFGF刺激的HRCEC细胞的增殖,其可能通过下调ERK1/2、P38和AKT蛋白分子的活化进而抑制细胞增殖。FGFR1-Fc抑制细胞周期进程,阻断G0/G1期细胞增殖。FGFR1-Fc抑制bFGF刺激的HRCEC细胞迁移,其可能通过抑制STAT3的活化水平下调迁移相关蛋白的表达而抑制血管生成。FGFR1-Fc和Razumab可抑制视网膜新生血管,两者具有协同作用。结论FGFR1-Fc抑制bFGF刺激的HRCEC增殖、迁移和血管生成。FGFR1-Fc与抗VEGF药物具有协同作用。

Objective To investigate the inhibitory effects of FGFR1-Fc on the proliferation,migration and angiogenesis of human retinal microvascular endothelial cell(HRCEC)stimulated by bFGF.To validate the inhibitory effects in oxygen-induced retinal neovascularization(OIR)mice model,and preliminarily study the synergistic effect of FGFR1-Fc and anti-VEGF drugs.Methods HRCEC were cultured in vitro and cells were treated with bFGF and different concentrations of FGFR1-FC.MTT assay was used to determine cell activity,scratch assay was used to detect cell migration ability,flow cytometry was used to detect cell cycle,and Western blotting was used to ascertain protein expression levels.OIR mice were used as animal models to observe the state of retinal blood vessels and calculate the area of retinal non-perfusion area.The extent of neovascularization was evaluated by counting the number of intravitreal vascular nuclei,which were defined as the nuclei of cells that extended beyond the inner limiting membrane of the retina into the vitreous vio Hematoxylin-eosin staining(HE).Results FGFR1-Fc inhibited bFGF-stimulated HRCEC proliferation,and suppressed p38,ERK1/2 and AKT phosphorylation,thus inhibitd the cell proliferation stimulated by bFGF.FGFR1-Fc inhibited cell cycle progression and blocked cell proliferation in G0/G1 phase.FGFR1-Fc inhibited bFGF-stimulated HRCEC migration,and FGFR1-Fc might down-regulate the expression of migration-related proteins by inhibiting the activation level of STAT3,and ultimately inhibited angiogenesis.FGFR1-Fc and Razumab could inhibit retinal neovascularization,and had a certain synergistic effect.Conclusion FGFR1-Fc inhibits bFGF-stimulated HRCEC proliferation,migration,and angiogenesis.FGFR1-Fc and anti-VEGF drugs have a synergistic effect.