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【摘要】目的研究诱导型一氧化氮合成酶（iNOS）和血管内皮生长因子（VEGF） 在碱烧伤后兔角膜新生血管（CNV）形成中的作用及其机制。方法用氢氧化钠滤纸片烧伤兔角膜建立新生血管动物模型，根据不同处理因素将实验兔分为三个实验组和一个对照组，每组12只。三个实验组结膜下注射不同剂量的三氧化二砷；对照组结膜下注射0.9%氯化钠。在裂隙灯下查看碱烧伤后角膜大体情况，计算角膜新生血管的面积。分别在兔角膜碱烧伤后7天、14天、28天，行兔角膜组织HE染色及免疫组化法检测iNOS和VEGF。 结果各实验组角膜新生血管面积均小于对照组 （P<0.05或0.01）；实验组角膜比对照组更光滑透明；角膜iNOS和VEGF表达情况：各组碱烧伤后第7天开始增高，14天时达到峰值，随后逐渐降低，并且对照组的表达明显高于实验组。结论兔角膜碱烧伤后CNV形成与iNOS和VEGF密切相关；三氧化二砷对CNV形成具有一定抑制作用。
【关键词】一氧化氮合成酶；血管内皮生长因子；三氧化二砷；角膜新生血管
中图分类号：R774.1 文献标识码：ADOI：10.3969/j.issn.10031383.2015.04.011
【Abstract】ObjectiveTo study effect and mechanism of inducible nitric oxide synthase （iNOS） and vascular endothelial growth factor（VEGF） in corneal neovascularization （CNV） after corneal alkali burn.MethodsAnimal models of neovascularization were established by burning rabbits corneas with sodium hydroxide filter papers.According to different kinds of treatments，experiment rabbits were divided into three experimental groups and one control group，with 12 rabbits in each group.The three experimental groups were given different doses of As2O3 by subconjunctival injection，while the control group were treated with 0.9% sodium chloride by subconjunctival injection.Corneas after alkali burn were observed under slit lamp，and areas of CNV were calculated.Rabbit corneal tissue HE staining and immunohistochemistry method were used to measure iNOS and VEGF 7，14 and 18 days after corneal alkali burn，respectively.Results Areas of CNV of all experimental groups were smaller than that of the control group（P<0.05 or 0.01），and the corneas of the experimental groups were more smooth and transparent than that of the control group.Expressions of iNOS and VEGF in corneas of all groups began to increase on the 7th day and reached the peak on the 14th day after alkali burn，then decreased gradually.And the expressions of the control group were significantly higher than those of the experimental groups.ConclusionCNV formation after corneal alkali burn is closely related with iNOS and VEGF.Besides，As2O3 has some inhibitory effect on CNV formation.
【Key words】iNOS；VEGF；As2O3；CNV
生理状态下角膜透明无血管，处于免疫赦免区之中，这是保持角膜正常生理功能的先决条件。在某些病理状态如炎症、碱烧伤、移植等时，会出现角膜新生血管。有研究表明，诱导型一氧化氮合成酶（inducible nitric oxide synthase，iNOS）在眼内含量丰富，角膜上皮和内皮均含有iNOS，其促进生成一氧化氮（NO），NO则能促进新生血管的形成[1]，此外iNOS对血管内皮生长因子（vascular endothelial growth factor，VEGF）具有一定调控作用[2～3]，VEGF是新生血管发生发展过程中重要的影响因子，因此在角膜新生血管形成机制的研究中该因子成为研究热点。目前，对于角膜新生血管的治疗非常棘手，至今还没有特效药物以及特效的治疗方法。近年来研究发现，作为抗肿瘤药物的三氧化二砷具有抑制新生血管的作用[4]。本研究应用氢氧化钠滤纸片烧伤兔角膜建立新生血管动物模型，结膜下注射三氧化二砷溶液，观察兔角膜碱烧伤后角膜新生血管生成的情况，免疫组化染色检测iNOS、VEGF在角膜新生血管形成过程中的表达，探讨角膜新生血管的发生机制。 [5] Huo X，Li Y，Jiang Y，et al.Inhibition of ocular neovascularization by coinhibition of VEGFA and PLGF[J].Cell Physiol Biochem，2015，35（5）：17871796.
[6] Feng CW，Wang LD，Jiao LH，et al.Expression of p53，inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions：correlation with clinical features[J].BMC Cancer，2002，2：8.
[7] Ando A，Yang A，Nambu H，et al. Blockade of nitricoxide synthase reduces choroidal neovascularization[J].Mol Pharmacol，2002，62（3）：539544.
[8] JianXian Yu，Lin Cui，QiYi Zhang，et al.Expression of NOS and HIF1a in human colorectal carcinoma and implication in tumor angiogenesis[J].World J Gastroenterol，2006，12（29）：46604664.
[9] CassiniVieira P，Araújo FA，da Costa Dias FL，et al. iNOS Activity Modulates Inflammation，Angiogenesis，and Tissue Fibrosis in PolyetherPolyurethane Synthetic Implants[J].Mediators Inflamm，2015，2015：138461.
[10] Dulak J，Jozkowicz A，DembinskaKiec A，et al.Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells[J].Arterioscler Thromb Vasc Biol，2000，20（3）：659666.
[11] Lu DY，Liou HC，Tang CH，et al.Hypoxiainduced iNOS expression in microglia is regulated by the PI3kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor1alpha[J].Biochem Pharmacol，2006，72（8）：9921000.
[12] Dong J，Cheng M，Sun H.Function of inducible nitric oxide synthase in the regulation of cervical cancer cell proliferation and the expression of vascular endothelial growth factor[J].Mol Med Rep，2014，9（2）：583589.
[13] Chen WL，Ouyang KX，Li HG，et al.Expression of inducible nitric oxide synthase and vascular endothelial growth factor in ameloblastoma[J].J Craniofac surg.2009，20（1）：171175.
【关键词】一氧化氮合成酶；血管内皮生长因子；三氧化二砷；角膜新生血管
中图分类号：R774.1 文献标识码：ADOI：10.3969/j.issn.10031383.2015.04.011
【Abstract】ObjectiveTo study effect and mechanism of inducible nitric oxide synthase （iNOS） and vascular endothelial growth factor（VEGF） in corneal neovascularization （CNV） after corneal alkali burn.MethodsAnimal models of neovascularization were established by burning rabbits corneas with sodium hydroxide filter papers.According to different kinds of treatments，experiment rabbits were divided into three experimental groups and one control group，with 12 rabbits in each group.The three experimental groups were given different doses of As2O3 by subconjunctival injection，while the control group were treated with 0.9% sodium chloride by subconjunctival injection.Corneas after alkali burn were observed under slit lamp，and areas of CNV were calculated.Rabbit corneal tissue HE staining and immunohistochemistry method were used to measure iNOS and VEGF 7，14 and 18 days after corneal alkali burn，respectively.Results Areas of CNV of all experimental groups were smaller than that of the control group（P<0.05 or 0.01），and the corneas of the experimental groups were more smooth and transparent than that of the control group.Expressions of iNOS and VEGF in corneas of all groups began to increase on the 7th day and reached the peak on the 14th day after alkali burn，then decreased gradually.And the expressions of the control group were significantly higher than those of the experimental groups.ConclusionCNV formation after corneal alkali burn is closely related with iNOS and VEGF.Besides，As2O3 has some inhibitory effect on CNV formation.
【Key words】iNOS；VEGF；As2O3；CNV
生理状态下角膜透明无血管，处于免疫赦免区之中，这是保持角膜正常生理功能的先决条件。在某些病理状态如炎症、碱烧伤、移植等时，会出现角膜新生血管。有研究表明，诱导型一氧化氮合成酶（inducible nitric oxide synthase，iNOS）在眼内含量丰富，角膜上皮和内皮均含有iNOS，其促进生成一氧化氮（NO），NO则能促进新生血管的形成[1]，此外iNOS对血管内皮生长因子（vascular endothelial growth factor，VEGF）具有一定调控作用[2～3]，VEGF是新生血管发生发展过程中重要的影响因子，因此在角膜新生血管形成机制的研究中该因子成为研究热点。目前，对于角膜新生血管的治疗非常棘手，至今还没有特效药物以及特效的治疗方法。近年来研究发现，作为抗肿瘤药物的三氧化二砷具有抑制新生血管的作用[4]。本研究应用氢氧化钠滤纸片烧伤兔角膜建立新生血管动物模型，结膜下注射三氧化二砷溶液，观察兔角膜碱烧伤后角膜新生血管生成的情况，免疫组化染色检测iNOS、VEGF在角膜新生血管形成过程中的表达，探讨角膜新生血管的发生机制。 [5] Huo X，Li Y，Jiang Y，et al.Inhibition of ocular neovascularization by coinhibition of VEGFA and PLGF[J].Cell Physiol Biochem，2015，35（5）：17871796.
[6] Feng CW，Wang LD，Jiao LH，et al.Expression of p53，inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions：correlation with clinical features[J].BMC Cancer，2002，2：8.
[7] Ando A，Yang A，Nambu H，et al. Blockade of nitricoxide synthase reduces choroidal neovascularization[J].Mol Pharmacol，2002，62（3）：539544.
[8] JianXian Yu，Lin Cui，QiYi Zhang，et al.Expression of NOS and HIF1a in human colorectal carcinoma and implication in tumor angiogenesis[J].World J Gastroenterol，2006，12（29）：46604664.
[9] CassiniVieira P，Araújo FA，da Costa Dias FL，et al. iNOS Activity Modulates Inflammation，Angiogenesis，and Tissue Fibrosis in PolyetherPolyurethane Synthetic Implants[J].Mediators Inflamm，2015，2015：138461.
[10] Dulak J，Jozkowicz A，DembinskaKiec A，et al.Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells[J].Arterioscler Thromb Vasc Biol，2000，20（3）：659666.
[11] Lu DY，Liou HC，Tang CH，et al.Hypoxiainduced iNOS expression in microglia is regulated by the PI3kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor1alpha[J].Biochem Pharmacol，2006，72（8）：9921000.
[12] Dong J，Cheng M，Sun H.Function of inducible nitric oxide synthase in the regulation of cervical cancer cell proliferation and the expression of vascular endothelial growth factor[J].Mol Med Rep，2014，9（2）：583589.
[13] Chen WL，Ouyang KX，Li HG，et al.Expression of inducible nitric oxide synthase and vascular endothelial growth factor in ameloblastoma[J].J Craniofac surg.2009，20（1）：171175.