HIF在兔角膜碱烧伤后CNV生成中的作用研究

Study of HIF on corneal neovascularization after corneal alkali burn in rabbits

目的研究缺氧诱导因子(hypoxia inducible factor,HIF)以及诱导性一氧化氮合成酶(inducible nitric oxide synthase,iNOS)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在角膜碱烧伤后角膜新生血管(corneal neovascularization,CNV)形成中的作用及机制。方法 建立兔CNV动物模型,将其随机分为对照组和3个实验组,对照组结膜下注射生理盐水,实验组注射不同剂量的三氧化二砷。观察兔CNV的生长情况,并记录CNV的生长面积。用免疫组织化学方法检测兔角膜HIF、iNOS以及VEGF的表达。结果 对照组6 d、12 d、15 d、21 d、28 d CNV的面积分别为:(53.86±20.60)mm2、(87.21±25.80)mm2、(128.31±40.10)mm2、(114.42±29.40)mm2、(78.15±35.13)mm2。各实验组CNV面积均小于对照组,差异均有统计学意义(均为P<0.05)。免疫组织化学方法检测角膜HIF-1α、iNOS及VEGF蛋白的表达:VEGF表达集中在角膜上皮下基质,存在于血管内皮细胞、炎性细胞胞浆内,呈深黄色。在7 d有较高表达,14 d达到高峰,28 d时明显降低,且对照组明显高于实验组,各实验组的表达随三氧化二砷剂量的增加而减少。蛋白的表达与CNV面积呈正相关。结论 角膜碱烧伤后CNV的形成可能与HIF-1α有关。三氧化二砷通过抑制HIF-1α和iNOS、VEGF表达而抑制兔CNV的形成。

Objective To investigate the effects and mechanisms of hypoxia inducible factor （HIF） ,inducible nitric oxide syathase （iNOS） and vascular endothelial growth factor （VEGF） in rabbit corneal neovascularization （CNV） after alkaline burn. Methods CNV models were established, and randomly divided into control group and three experimental groups. Rabbits in control group were treated with subconjunctival injection of normal saline;Rabbits in experimental groups were treated with different doses of arsenic trioxide. Growth condition and areas were observed and recorded. Immunohistochemical method was used to detect expression of HIF, iNOS and VEGF in cornea. Results At 6 days, 12 days, 15 days, 21 days and 28 days, CNV areas were （53.86 ± 20.60） mm：, （ 87.21 ± 25.80 ） nun2, （ 128.31 ± 40. 10 ） mm2, （ 114.42 ± 29.40 ） mm2, （78.15 ± 35.13 ）mm2 in control group. CNV areas in each experimental group were smaller than that in control group （ all P 〈 0.05 ）. The expressions of HIF, iNOS and VEGF with intmunohistochemical method： expression of VEGF were mainly found in inferior matrix of corneal epithelium, vascular endothelial cells and inflammatory cells, and with dark yellow. The high expression was detected at 7 days, went to peak at 14 days and obviously decreased at 28 days. The expression in control group was obviously higher than that in experimental groups, and it decreased with the increase of arsenic trioxide. Conclusions After alkali burn,CNV may have relation with HIF-1α. And arsenic trioxide can inhibit the expression of HIF, iNOS and VEGF, thus play the inhibitive effect on CNV.