基质金属蛋白酶2、9和血管内皮生长因子在碱烧伤小鼠角膜组织中的表达

The expression of MMP2、MMP9 and VEGF in alkali-burn induced mouse corneal neovascularization

采取滤纸片放置法,用1mol/L的NaOH在小鼠角膜中央进行碱烧伤,建立动物模型。取烧伤前和烧伤后的小鼠角膜制备组织切片,用抗MMP2、抗MMP9和抗VEGF的抗体分别进行免疫组化染色,检测它们在碱烧伤前后的小鼠角膜组织中的蛋白表达情况;提取碱烧伤前和碱烧伤后不同时间点的小鼠角膜组织的总RNA并进行逆转录,以获得的逆转录产物作为模板,用特异的引物和探针进行实时定量PCR,分别检测MMP2、MMP9和VEGF在碱烧伤前后的小鼠角膜组织中转录水平的变化;提取碱烧伤前后小鼠角膜的蛋白组分,用明胶底物酶谱的方法检测其中MMP2和MMP9的活性变化。结果表明,浸透1mol/L的NaOH的滤纸小片,对小鼠角膜进行碱烧伤可以有效地诱导角膜新生血管形成,烧伤后3～4d就有明显的新生血管形成,至第8、9d左右开始退行。免疫组化结果显示,正常角膜中没有MMP9和VEGF表达,MMP2表达极低;碱烧伤后3种因子的表达均上升。实时定量PCR的结果显示MMP2在碱烧伤后表达升高,第3d达到最高随后下降;MMP9在正常角膜组织中没有表达,碱烧伤后第1d达到最高之后开始下降;VEGF在正常角膜中也没有表达,碱烧伤后第4d达到高峰之后开始下降。以上结果说明碱烧伤后的角膜组织中MMP2、MMP9和VEGF的表达均经历先上升后下降的变化。

Detect and analysis the expression change of MMP2, MMP9 and VEGF in mouse cornea before and after corneal alkali-burn. Mouse cornea was burned with NaOH (1 mol/L) saturated filter paper, the induction of angiogenesis was observed in the following day. The rabbit anti-mouse MMP2, rabbit anti-mouse MMP9 and rabbit anti-mouse VEGF were used in the immunohistochemistry stain. The standard immunohistochemistry stain procedure was performed. The Real-time PCR was performed to detect the mRNA change of MMP2, MMP9 and VEGF in alkali-burned mouse cornea. GAPDH were used here as inner standard. Gelatin-Zymography was performed to test the MMP activity in mouse cornea collected at different time points. The results showed the immunohistochemistry cannot detect MMP9 and VEGF signal in normal mouse cornea and the anti-MMP2 antibody only slight stain the epithelial of normal mouse cornea, while the MMP2, MMP9 and VEGF can by stain strongly in alkali-burned mouse cornea. Real-time PCR can hardly detect MMP9, VEGF expression in normal mouse cornea, and the MMP2 expression is very low and in alkali-burned mouse corneal the expression of MMP2, MMP9, VEGF were up-regulate immediate and then decrease gradually. The gelatin-zymograpy also shows increased activity of MMP in alkali-burned mouse cornea. After alkali burn the mouse corneal expression of MMP2, MMP9 and VEGF increased immediately and reach the peak, then decrease progressively, in accordance with the cornea wound healing and the developing and regress process of angiogenesis.