CD146在角膜新生血管形成中的动态表达及其意义

The dynamic expression of CD146 in corneal neovascularization and its significance

目的探讨CD146在角膜新生血管中的表达及其定位。方法8周龄SPF级健康雄性SD大鼠50只，采用随机数字表法将50只大鼠随机分为碱烧伤后1 d组、碱烧伤后3 d组、碱烧伤后7 d组、碱烧伤后14 d组和正常对照组，每组10只。采用NaOH碱烧伤法构建角膜新生血管模型，裂隙灯显微镜下观察碱烧伤后不同时间点角膜新生血管面积，随后制备角膜组织切片，采用逆转录PCR法检测CD146 mRNA的表达，Western blot法检测CD146蛋白的表达，采用免疫组织化学染色检测角膜组织中CD146的表达和定位。结果碱烧伤后3 d裂隙灯显微镜下可观察到角膜新生血管，碱烧伤后7 d角膜新生血管形成达高峰，随后逐渐消退。碱烧伤后1、3、7和14 d组角膜新生血管面积分别为（1.9±0.7）、（10.3±1.1）、（29.6±2.4）和（11.8±1.0）mm2，整体比较差异有统计学意义（F＝650.976，P＝0.000），其中碱烧伤后7 d组角膜新生血管面积最大，碱烧伤后1、3和14 d组与碱烧伤后7 d组比较，差异均有统计学意义（t＝－33.293、－20.475、20.744，均P＝0.000）。碱烧伤后1、3、7和14 d组CD146 mRNA的相对表达量分别为0.3±0.1、1.1±0.2、3.5±0.4和1.3±0.3，各组蛋白的相对表达量分别为0.2±0.1、1.4±0.2、4.1±0.5和1.3±0.2，各组间整体比较差异均有统计学意义（F＝1 176.920，P＝0.000；F＝233.127，P＝0.000），其中碱烧伤后7 d组CD146 mRNA和蛋白的相对表达量均最高，碱烧伤后1、3和14 d组与碱烧伤后7 d组比较，差异均有统计学意义（mRNA：t＝－58.109、－33.725、31.006，均P＝0.000；蛋白：t＝－59.873、－38.762、39.153，均P＝0.000）。免疫组织化学染色结果显示，CD146于碱烧伤后7 d在角膜新生血管内皮细胞中高表达，形成管腔结构，而碱烧伤后14 d在成熟的角膜新生血管中仅有微量CD146表达。论CD146与角膜新生血管形成关系密切，有希望作为抗新生血管治疗新的靶点。

ObjectiveTo investigate the expression and localization of CD146 in corneal neovascularization.MethodsFifty SPF male SD rats with the age of 8 weeks were randomly divided into 1 day group, 3 days group, 7 days group, 14 days group and normal control group by using random number table method, 10 rats for each group.Corneal neovascularization models were established by sticking the filter papers with NaOH on the central corneas.The area of corneal neovascularization was observed at different time points by slit lamp microscopy.The expression of CD146 mRNA and protein were detected by reverse transcription PCR and Western blot, respectively.The location of CD146 expression was observed by using immunohistochemical staining method.The animal feeding and use was in accordance with the standards set by the ARVO, and the experiment was approved by the Ethic Committee for Experimental Animal of Affiliated Hospital of Xuzhou Medical University （2016012）.ResultsCorneal neovascularization occurred at 3 days and peaked at 7 days after alkali burn, then gradually subsided.The area of corneal neovascularization was （1.9±0.7）, （10.3±1.1）, （29.6±2.4） and （11.8±1.0）mm2 in 1 day, 3 days, 7 days and 14 days after alkali burn, respectively, the overall difference was statistically significant （F=650.976, P=0.000）, and the area of corneal neovascularization in 1 day group, 3 days group, 14 days group was significantly shrinked compared with that in the 7 days group after alkali burn, with significant differences between them（t=-33.293, -20.475, 20.744, all at P=0.000）, while there was no significant differences between the other groups （all at P〉0.05）. The relative expression levels of CD146 mRNA in the 1 day, 3 days, 7 days and 14 days group after alkali burn was 0.3±0.1, 1.1±0.2, 3.5±0.4 and 1.3±0.3, and the relative expression levels of CD146 protein was 0.2±0.1, 1.4±0.2, 4.1±0.5 and 1.3±0.2, respectively, the overall differences between the four groups were statistically significant （F=1 176.920, P=0.000; F=233.127, P=0.000）, and the relative expression levels of CD146 mRNA and protein in the 1 day, 3 days, 7 days and 14 days group were lower than those in the 7 days group after alkali burn, with significant differences between them （mRNA： t=-58.109, -33.725, 31.006; all at P=0.000.protein： t=-59.873, -38.762, 39.153, all at P=0.000）. Immunohistochemical results showed that CD146 was highly expressed in corneal neovascular endothelial cells at 7 days after alkali burn, forming lumen structure, while only weak expression of CD146 could be detected in mature corneal neovascularization at 14 days after modeling.ConclusionsCD146 is closely related to corneal neovascularization formation, and it is promising as a new target for treatment.