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

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。

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

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(P>0.05).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(P<0.05),D405 in 10 ng/mL VEGF group was significantly lower than that in control group(P<0.05),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(P<0.05),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(P<0.05),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(P<0.05).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.