上海交通大学学报（医学版）

›› 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

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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