胰岛素样生长因子结合蛋白相关蛋白-1抑制视网膜血管新生的机制研究

Mechanism of inhibitory effect of insulin-like growth factor binding protein-related protein 1 on retinal angiogenesis

目的探讨胰岛素样生长因子结合蛋白相关蛋白-1(IGFBP-rP1)抑制血管内皮细胞生长因子(VEGF)体外诱导视网膜新生血管形成的机制。方法猕猴视网膜/脉络膜血管内皮细胞株(RF/6A)扩增培养、血清饥饿培养24 h后,分为对照组及50、100、200 ng/mL IGFBP-rP1干预组进行干预,流式细胞术检测细胞凋亡率的变化;分为对照组,10 ng/mL VEGF干预组,50、100、200 ng/mL IGFBP-rP1+10 ng/mL VEGF干预组进行干预,免疫细胞化学染色检测细胞中B-Raf蛋白表达,分光光度法检测细胞中Caspase 3活性的变化。结果流式细胞仪检测显示,50、100、200 ng/mL IGFBP-rP1干预组RF/6A细胞凋亡率与对照组比较差异无统计学意义(P>0.05)。免疫细胞化学染色检测与Caspase 3活性检测显示,与对照组比较,10 ng/mL VEGF干预组RF/6A细胞细胞质平均吸光度显著升高、D405值显著降低(均P<0.05);与10 ng/mL VEGF干预组比较,50、100、200 ng/mL IGFBP-rP1+10 ng/mL VEGF干预组RF/6A细胞细胞质平均吸光度显著降低、D405值显著升高(均P<0.05),且各组间比较差异均有统计学意义(均P<0.05)。结论 IGFBP-rP1通过干预B-Raf蛋白表达,上调细胞Caspase 3活性,对抗VEGF的促视网膜血管新生效应,发挥抑血管新生因子的负向调节作用。

Objective To investigate the mechanism of inhibitory effect of insulin-like growth factor binding protein-related protein（IGFBP-rP1） on vascular endothelial growth factor（VEGF）-induced retinal angiogenesis in vitro.MethodsRetina-choroid endothelial cell line（RF/6A） of macaque was cultured and starved for 24 h.Then RF/6A cells were divided into control group and 50,100 and 200 ng/mL IGFBP-rP1 groups,and flow cytometry was used to detect the apoptotic ratios of RF/6A cells.The starved RF/6A cells were also divided into control group,10 ng/mL VEGF group and 50,100 and 200 ng/mL IGFBP-rP1＋10 ng/mL VEGF groups,the expression of B-Raf protein was detected with immunocytochemical staining,and the activation of Caspase 3 was determined by spectrophotometric method.Results Flow cytometry revealed that there was no significant difference in apoptotic ratios of RF/6A cells between control group and 50,100 and 200 ng/mL IGFBP-rP1 groups（P0.05）.Immunocytochemical detection and Caspase 3 activation detection demonstrated that the average optical density of RF/6A cells in 10 ng/mL VEGF group was significantly higher than that in control group（P0.05）,D405 in 10 ng/mL VEGF group was significantly lower than that in control group（P0.05）,the average optical density of RF/6A cells in 50,100 and 200 ng/mL IGFBP-rP1＋10 ng/mL VEGF groups was significantly lower than that in 10 ng/mL VEGF group（P0.05）,D405 in 50,100 and 200 ng/mL IGFBP-rP1＋10 ng/mL VEGF groups was significantly higher than that in 10 ng/mL VEGF group（P0.05）,and there were significant differences in average optical density of RF/6A cells and D405 among 50,100 and 200 ng/mL IGFBP-rP1＋10 ng/mL VEGF groups（P0.05）.ConclusionIGFBP-rP1 inhibits the biologic proceedings induced by VEGF during retinal angiogenesis by intervention of expression of B-Raf protein and upregulation of Caspase 3 activity.