实验性脉络膜新生血管中Rapl、鸟苷三磷酸-Rap1、血管内皮生长因子及β-连环蛋白的表达研究

Expression of Rapl, guanosine triphosphate Rapl, vascular endothelial growth factor and β-catenin in experimental choroidal neovascularization

目的观察实验性脉络膜新生血管（CNV）中Rap1、鸟苷三磷酸-Rap1（GTP-Rap1）、血管内皮生长因子（VEGF）及β-连环蛋白（β-catenin）的表达。方法42只棕色挪威大鼠随机分为空白对照组、模型组，分别为7、35只；均双眼入组。模型组大鼠氪离子激光光凝建立CNV模型。光凝后3、7、14、21、28 d行荧光素眼底血管造影（FFA）、脉络膜血管铺片检查，观察光凝后不同时间大鼠荧光素渗漏程度以及CNV面积的变化。蛋白免疫印迹法（Western blot）、实时荧光定量聚合酶链反应（RT-PCR）检测CNV中Rap1、GTP-Rap1、VEGF、β-catenin蛋白和mRNA表达。结果FFA检查结果显示，光凝后14 d，光凝斑出现大片圆盘状荧光素渗漏。激光共聚焦显微镜观察发现，与光凝后7 d时CNV面积比较，光凝后14、21、28 d时CNV面积增加，差异均有统计学意义（t=3.725、5.532、3.605，P＜0.05）。Western blot检测结果显示，与空白对照组比较，光凝后不同时间点，CNV中Rap1蛋白相对表达量差异均无统计学意义（P=0.156）；GTP-Rap1蛋白相对表达量显著降低，VEGF、β-catenin蛋白相对表达量明显增高，差异均有统计学意义（P=0.000）。RT-PCR检测结果显示，与空白对照组比较，光凝后不同时间点，CNV中Rap1 mRNA相对表达量差异无统计学意义（P=0.645）；β-catenin mRNA相对表达量差异均有统计学意义（P=0.000）。7、14、21、28 d，GTP-Rap1、VEGF mRNA相对表达量差异均有统计学意义（P=0.000）。结论实验性CNV中GTP-Rap1表达较正常大鼠明显减少。

ObjectiveTo observe the expression of Rap1, guanosine triphosphate-Rap1 （GTP-Rap1）, vascular endothelial growth factor （VEGF） and β-catenin in experimental choroidal neovascularization （CNV）.MethodsForty-two brown Norwegian rats were randomly divided into a blank control group （7 rats） and a model group （35 rats）. Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography （FFA） and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction （RT-PCR） were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV.ResultsThe results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant （t=3.725, 5.532, 3.605;P〈0.05）. Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups （P=0.156）. Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group （P=0.000）. The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups （P=0.645）, but there were significant difference in the relative expression of β-catenin mRNA （P=0.000）. At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups （P=0.000）.ConclusionsThe expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.