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CXCL12/CXCR4轴调节小鼠角膜新生血管的实验研究

杜玲玲  杜海涛  刘平   

  1. 150001 哈尔滨医科大学附属第一医院眼科
  • 收稿日期:2017-04-25 出版日期:2018-11-25 发布日期:2018-12-11
  • 通讯作者: 刘平,Email:pingliu53@126.com E-mail:pingliu53@126.com
  • 基金资助:

    黑龙江省博士后科研启动金(LBH-Z17101);哈尔滨医科大学创新科学研究资助项目(2017LCZX03)

CXCL12/CXCR4 axis regulates neovascularization in sutured corneas in mice

DU Ling-ling, DU Hai-tao, LIU Ping   

  1. The First Affiliated Hospital of Harbin Medical University, Harbin 150001,China
  • Received:2017-04-25 Online:2018-11-25 Published:2018-12-11
  • Contact: LIU Ping, Email: pingliu53@126.com E-mail:pingliu53@126.com

摘要:

目的 观察CXCL12/CXCR4轴与VEGF-A家族及角膜新生血管之间的关系。设计 实验研究。研究对象 24只小鼠。方法 将小鼠分为三组,每组8只。采用标准三针缝线法诱导小鼠出现角膜新生血管,1组为缝线诱导的角膜新生血管模型组,2组为缝线联合平衡盐溶液结膜下注射组,3组为缝线联合AMD 3100结膜下注射组。观察7天后角膜新生血管情况,免疫组化方法检测 CXCR4表达,荧光定量PCR、蛋白印迹法检测CXCL12/ CXCR4以及VEGF-A、VEGFR-1的表达,免疫荧光分析检测新生血管侵入的面积。主要指标 CXCR4表达,CXCL12/ CXCR4以及VEGF-A、VEGFR-1的表达,角膜新生血管面积。结果 在缝线模型组,CXCL12(0.40±0.01 μg)、CXCR4(0.40±0.03 μg)、VEGF-A(0.99+0.39 μg)、VEGFR-1 (0.91+0.45 μg)在mRNA和蛋白水平的表达均明显升高。在结膜下注射AMD3100组中CXCL12(0.20±0.02 μg)、CXCR4(0.28±0.07 μg)、VEGF-A(0.64+0.20 μg)及VEGFR-1 (0.64+0.25 μg)在mRNA和蛋白水平的表达降低。CXCL12/CXCR4的表达增多与免疫荧光显示的新生血管面积增大一致(1.00±0.22)。结论 CXCL12/CXCR4通过VEGF-A参与角膜新生血管生成,AMD3100可望用于控制角膜新生血管形成。

关键词: 新生血管形成, CXCL12/CXCR4, 血管内皮生长因子

Abstract:

Objective To determine the role of chemokine (C-X-C motif) ligand 12 (CXCL12)/chemokine (C-X-C motif) receptor 4 (CXCR4) in inflammatory corneal hemangiogenesis, and to study the relationship between VEGF-A family and CXCL12 / CXCR4 in the corneal neovascularization. Design Experimental study. Participants Twenty-four mice. Methods Corneal hemangiogenesis was induced by placing three 11-0 nylon sutures in the corneal intrastromal. Mice were divided into three groups (8 mice in saline control group, AMD3100 subconjunctival injection group and suture model group each). Immunohistochemical method was used to investigate the CXCR4 expression. Fluorescence quantitative PCR and Western blot were used to investigate the expression of CXCL12 / CXCR4 and VEGFA family. Immunofluorescence assay was used to measure the area of new blood vessels invasion. Main Outcome Measures The expression of CXCR4, CXCL12/CXCR4 and VEGFA family, and the area of new blood vessels invasion. Results CXCL12/CXCR4 mRNA and protein expression levels increased markedly in suture-induced corneal neovascularization (CXCL12: 0.40±0.01 μg, CXCR4: 0.40±0.03 μg, VEGF-A: 0.99+0.39 μg, VEGFR-1: 0.91+0.45 μg) and decreased in the AMD3100 group (CXCL12: 0.20±0.02 μg, CXCR4: 0.28±0.07 μg, VEGF-A: 0.64+0.20 μg, VEGFR-1: 0.64+0.25 μg). Hemangiogenesis was captured in immunofluorescence images and the area was shown to be markedly increased in the suture model group (1.00±0.22),  which was consistent with the increasing expression of the CXCL12/CXCR4. Conclusion CXCL12/CXCR4 regulates hemangiogenesis in suture-induced corneal hemangiogenesis. AMD3100 may be a novel therapeutic target for the prevention of corneal neovascularization.

Key words: neovascularization, CXCR4/CXCL12, vascular endothelial growth factor