Chen Peng, Yin Hongmei, Wang Yao, Mi Jing, He Wenxiao, Xie Lixin, Wang Yiqiang
Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China.
Mol Vis. 2010 Feb 27;16:310-9.
To determine the effectiveness of multigene-based anti-angiogenic gene therapies for experimental murine corneal neovascularization (corneal NV).
Recombinant retroviral vectors encoding murine endostatin (mEndo), murine-soluble vascular endothelial growth factor receptor-2 (msFlk-1), or murine-soluble Tie2 (msTie2) were constructed and packaged in PT67 cells. Viral titers were determined by infection of NIH3T3 cells. Expressions of mEndo, msFlk-1, and msTie2 were confirmed by reverse transcription PCR. The 3-(4,5-Dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to estimate the effect of mEndo, msFlk-1, or msTie2 on the proliferation of human umbilical vein endothelial cells, and the scarification test was used to measure the migration of the cells. Seventy C57Bl/6 mice were subjected to the induction of chemical-burn corneal NV and tested for efficacy of gene therapy. Gene therapy was performed by subconjunctival injection of viral preparations and its effect was evaluated by scoring corneal NV.
The recombinant virus-producing cell lines expressing mEndo, msFlk-1, and msTie2 were constructed successfully. Overexpression of these putative anti-angiogenic proteins inhibited the proliferation and migration of human umbilical vein endothelial cells in vitro. In the murine corneal NV model, subconjunctival injection of the retroviral particles of mEndo and msFlk-1 showed the most significant inhibition of corneal NV.
Gene therapy with the recombinant retroviral vector-hosted mEndo and msFlk-1 gene effectively inhibited corneal NV induced by alkaline burn. The combination of multiple anti-angiogenic genes might be necessary for effective therapy of corneal NV, although each of these pathways makes a potential target for the treatment of this disease.
确定基于多基因的抗血管生成基因疗法对实验性小鼠角膜新生血管形成（角膜NV）的有效性。
构建编码小鼠内皮抑素（mEndo）、小鼠可溶性血管内皮生长因子受体-2（msFlk-1）或小鼠可溶性Tie2（msTie2）的重组逆转录病毒载体，并在PT67细胞中包装。通过感染NIH3T3细胞测定病毒滴度。通过逆转录PCR确认mEndo、msFlk-1和msTie2的表达。使用3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2H-四唑溴盐（MTT）法评估mEndo、msFlk-1或msTie2对人脐静脉内皮细胞增殖的影响，并使用划痕试验测量细胞的迁移。70只C57Bl/6小鼠接受化学烧伤诱导的角膜NV，并测试基因治疗的疗效。通过结膜下注射病毒制剂进行基因治疗，并通过对角膜NV进行评分来评估其效果。
成功构建了表达mEndo、msFlk-1和msTie2的重组病毒生产细胞系。这些假定的抗血管生成蛋白的过表达在体外抑制了人脐静脉内皮细胞的增殖和迁移。在小鼠角膜NV模型中，结膜下注射mEndo和msFlk-1的逆转录病毒颗粒对角膜NV的抑制作用最为显著。
用重组逆转录病毒载体携带的mEndo和msFlk-1基因进行基因治疗可有效抑制碱性烧伤诱导的角膜NV。尽管这些途径中的每一个都可能成为治疗这种疾病的潜在靶点，但多种抗血管生成基因的组合可能是有效治疗角膜NV所必需的。
