›› 2012, Vol. 32 ›› Issue (2): 133-.doi: 10.3969/j.issn.1674-8115.2012.02.002

• 论著(基础研究) • 上一篇    下一篇

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

孙 涛, 朱弼珺, 许 迅, 苏 莉, 顾 青, 许 琳   

  1. 上海交通大学附属第一人民医院眼科, 上海 200080
  • 出版日期:2012-02-28 发布日期:2012-02-28
  • 通讯作者: 许 迅, 电子信箱: dr_xuxun@yahoo.com.cn。
  • 作者简介:孙 涛(1978—), 男, 主治医师, 博士;电子信箱: drsuntao@yeah.net。
  • 基金资助:

    国家自然科学基金重点项目(30930097);国家自然科学基金青年基金(30801269);上海交通大学医学院博士创新基金(BXJ0935);上海市眼底病重点实验室开放课题基金(07Z22911)

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

SUN Tao, ZHU Bi-jun, XU Xun, SU Li, GU Qing, XU Lin   

  1. Department of Ophthalmology, the First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China
  • Online:2012-02-28 Published:2012-02-28
  • Supported by:

    State Key Program of National Natural Science Foundation of China, 30930097;National Natural Science Foundation for Young Scholars of China, 30801269;Doctoral Innovation Fund of Shanghai Jiaotong University School of Medicine, BXJ0935;Shanghai Key Laboratory for Ocular Fundus Diseases Foundation, 07Z22911

摘要:

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。

关键词: 胰岛素样生长因子结合蛋白相关蛋白-1, 血管内皮细胞生长因子, RF/6A细胞, B-Raf, Caspase 3

Abstract:

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. Methods Retina-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). Conclusion IGFBP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.

Key words: insulin-like growth factor binding protein-related protein 1, vascular endothelial growth factor, RF/6A cell, B-Raf, Caspase 3