上皮缺损诱导角膜新生血管小鼠模型中VEGF与sFlt-1变化趋势的实验研究

Changes of VEGF and sFlt-1 levels in murine corneal neovascular models in mice induced by epithelium defect

目的观察小鼠角膜上皮缺损与角膜新生血管发生发展的关系,初步探讨新生血管发生发展过程中血管内皮生长因子(vascular endothelial growth factor,VEGF)与血管内皮生长因子可溶性受体(soluble VEGF receptor-1,sFlt-1)含量的变化趋势。方法正常成年雄性昆明小鼠294只,平均分为2组,分别接受角膜缘损伤加角膜上皮刮除(A组)和单纯角膜上皮刮除(B组)2种处理,干预后1、3、5、7、10、14d取角膜,分别行苏木素-伊红(hematoxylin eosin,HE)及过碘酸-希夫染色,铺片,免疫荧光标记血管内皮细胞,非变性蛋白印迹分析各时间点结合态和游离态VEGF蛋白含量,实时定量聚合酶链反应分析对应时间点sFlt-1信使核糖核酸(message ribonucleic acid,mRNA)的表达强度。结果两组均成功诱导角膜新生血管。除第14天外,A组各时间点新生血管面积均高于B组,但前者上皮修复延迟,早期炎性细胞浸润程度亦较高,晚期角膜部分结膜化。A组各时间点VEGF总蛋白及游离蛋白含量均明显高于B组;sFlt-1 mRNA表达于早中期下降而后期上升。结论持续一定时间的角膜上皮缺损可相对减少sFlt-1的表达,增加游离VEGF,诱导血管生成;角膜缘损伤通过加重上皮缺损程度,增加血管形成。

Objective To observe the relationship between epithelium defect and corneal neovascularization and to investigate the changes of vascular endothelial growth factor（VEGF） and soluble VEGF receptor-1 （ sFlt-1 ） in murine models induced by corneal epithelium defect. Methods Two hundred and ninety-four normal adult kunming mice were divided into two groups. Superfacial limbal epithelium debridement combined with complete corneal epithelium debridement was induced in group A and complete epithelium debridement in group B. Corneas were excised at day 1,3,5,7,10 and 14 respectively. The epithelium restoration and the density of inflammatory cells and goblet cells were observed in hematoxylin eosin and periodic acid-Schiff sections. The time-dependent area of neovessels was analyzed by immunofluorescenee of corneal flatmounts. Content of sFlt-1 message ribonucleic acid mRNA and VEGF including bound and free form was assessed with real-time polymerase chain reaction and non-reducing western blot. Results Corneal neovascularization was successfully induced in both groups. The area of neovascularization of group A was significantly larger than that of group B except at day 14. The corneal epithelium restoration was delayed in group A. The density of inflammatory cells was higher in the very early stage and the goblet cells appeared in group A at the late phase. The content of VEGF of both form in group A was throughout, much higher than that of group B. Expression of sFlt-1 mRNA was downregulated at the early stage,but upregulated later. Conclusions Persistent epithelium defect relatively reduced the expression of sFlt-1, increased the content of free VEGF protein, and finally promoted corneal neovascularization. Damage of corneal limbus aggravated epithelium defect and increased the area of blood vessels. （Chin J Ophthalmol and Otorhinolaryngo1,2009,9 ：80- 82）