碱烧伤后小鼠角膜新生血管表达vasohibin mRNA及其蛋白

Neovascularized vessels express vasohibin mRNA and protein in alkaline burned mice cornea

目的:探讨碱烧伤后的小鼠角膜新生血管(corneal neovascularization,CNV)是否表达vasohibin mRNA及其蛋白。方法:15只小鼠采用碱烧伤的方法制备角膜新生血管模型。6只小鼠为正常对照组。分别于烧伤后1、3、6、9d裂隙灯显微镜观察CNV情况并拍照,以NIH ImagJ图像软件分析角膜照片,计算新生血管的面积比(新生血管占全角膜的比值)。分别在这四个不同时间点提取烧伤组各3个角膜的总mRNA,另取3个正常对照组角膜作为对照,用半定量逆转录酶链式反应(RT-PCR)检测vasohibin mRNA在CNV中的表达水平。在烧伤后9d采用免疫荧光染色法检测vasohibin蛋白在烧伤和正常角膜组织中的表达异同。结果:vasohibin蛋白在烧伤组角膜中的新生血管内皮呈强荧光信号,而在正常角膜组织中没有荧光信号;碱烧伤后1、3、6和9d,新生血管面积比分别为0%、(12.72±1.95)%、(30.45±5.40)%、(53.09±9.14)%,vasohibin mRNA相对GAPDH mRNA的表达量为2.11±0.32、2.39±0.76、3.72±0.54 and 8.94±1.21。在观察期内,新生血管面积比值和vasohibin mRNA的表达水平逐渐升高,二者呈现明显的相关性(r=0.930,P<0.05)。结论:碱烧伤后小鼠角膜新生血管表达vasohibin mRNA及其蛋白,但正常角膜组织中不表达。Vasohibin蛋白表达与角膜新生血管的形成关系密切。

Objective：To investigate whether the expression of vasohibin mRNA and its protein exists in the chemical burned mouse cornea. Methods ：Corneal neovasularization （CNV） was induced by alkaline bums in 15 mice. Six mice with normal corneas were taken as controls. The mice eyes were examined by flit-lamp microscopy ,the area of CNV was analyzed by NIH ImagJ software and the percentage of neovaseulafized area to entire cornea was calculated at 1,3,6, and 9 days after chemical injury. Total RNA from three mice corneas each was extracted at the four checkpoints after injury, while 3 normal corneas as controls. The levels of vasohibin mRNA expression were examined by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） for every check time and its protein was detected by indirect immanofluoresoent staining in burned and normal corneas at day 9 after bums. Results：Positive signals were noted in the burned corneas, but not in the normal mice corneas by immunostaining.The percentages of neovaseularized area to entire cornea were 0%, （12.72±1.95）%, （30.45±5.40）%, （53.09±9.14）%, and the quantities of relative expression of vasohibin mRNA to GAPDH mBNA were 2.11±0.32,2.39±0.76,3.72±0.54 and 8.94±1.21 at 1,3,6,and 9 days after corneal bums. There is a strong correlation statistically between the degree of corneal neovascularization and vasohibin mRNA expression （r=0.930,P〈0.05）, beth of them gradually increased over observation time. Conelusion：Vasohibin mRNA and protein expressed in neovaseularized vessels in alkaline burned mice cornea,but not in normal cornea. The strength of its expression is closely relative to the degree of corneal neovaseularization.