血管生成素1对大鼠脉络膜新生血管的治疗作用及其初步机制

Therapeutic effect and mechanism of angiopoietin 1 on choroidal neovascularization

目的 探究血管生成素1(Ang1)对大鼠脉络膜新生血管(CNV)的治疗作用及其可能机制。方法 选取30只6~8周龄挪威(BN)大鼠,雌雄不限,随机分为正常组、模型组、Ang1治疗组,每组10只。正常组不做处理;其余两组采用多波长氪激光对BN大鼠双眼进行CNV造模,于造模后14 d进行眼底荧光素血管造影检查。造模成功后1 d,Ang1治疗组玻璃体腔注射200μg/L的Ang1 20μl,其余两组注射等量生理盐水;注药后10 d进行眼底荧光素血管造影检查,使用FITC标记葡聚糖(FITC-dextran)心脏灌注法进行脉络膜铺片测量CNV面积,采用苏木精-伊红(HE)染色观察大鼠视网膜-脉络膜结构变化;取大鼠视网膜-脉络膜-巩膜复合体,采用Western blotting检测Ang1、Rap1、小分子G蛋白Rap1(GAPRap1)、血管内皮-钙黏蛋白(VE-cadherin)的表达情况。取对数生长期大鼠脉络膜血管内皮细胞(RCVECs),用血管内皮生长因子(VEGF)刺激培养24 h后,分为阴性对照组(siRNA-NC组)、GAPRap1小干扰RNA组(GAPRap1-siRNA组)、GAPRap1-siRNA+Ang1组。GAPRap1-siRNA组及GAPRap1-siRNA+Ang1组转染GAPRap1-siRNA试剂,siRNA-NC组细胞转染siRNA-NC试剂,转染6 h后,在GAPRap1-siRNA+Ang1组中加入200μg/L Ang1,继续培养24 h后提取细胞蛋白,采用Western blotting检测细胞中GAPRap1、Rap1、VE-cadherin的表达情况,免疫荧光染色检测转染后细胞中VE-cadherin的表达水平。结果 与正常组比较,模型组大鼠新生血管渗漏面积、脉络膜损伤程度明显升高,脉络膜中GAPRap1、VE-cadherin蛋白表达明显降低(P<0.05),Rap1蛋白表达无明显变化(P>0.05);与模型组比较,Ang1治疗组大鼠新生血管渗漏面积、脉络膜损伤程度明显降低,脉络膜与细胞中GAPRap1、VE-cadherin蛋白表达明显升高(均为P<0.01),Rap1蛋白表达无明显变化(P>0.05)。脉络膜组织中,Ang1蛋白在Ang1治疗组的表达明显高于其余两组(P<0.01)。细胞实验中,GAPRap1-siRNA组GAPRap1与VE-cadherin的表达水平较GAPRap1-siRNA+Ang1组及siRNA NC组明显降低(P<0.01),而Rap1表达无明显变化(P>0.05)。免疫荧光染色检测结果显示,GAPRap1-siRNA组VE-cadherin表达水平较GAPRap1-siRNA+Ang1组及siRNA NC组明显降低(P<0.001)。结论 Ang1可减少大鼠CNV的渗漏,对CNV生长具有一定抑制作用,其机制可能与通过GAPRap1-VEcadherin途径加强细胞黏附作用有关。

Objective To investigate the effect and potential mechanism of angiopoietin 1(Ang1)on choroidal neovascularization(CNV)of rats.Methods A total of 30 Norwegian(BN)rats aged 6-8 weeks were randomized into three groups(n=10/group):normal group,model group,and Ang1 treatment group.The normal group was not processed,but the other two groups used multi-wavelength krypton laser to model the eyes of BN rats.We then performed fundus fluorescein angiography 14 days post-surgery.After confirming the success of the surgery,on the next day,the Ang1 treatment group received 200μg/L Ang120μl through vitreous cavity injection,while the other two groups received an equal volume of saline.After another 10-day,we performed Fundus fluorescein angiography examination,measured the CNV area through choroidal patching using FITC-labeled dextran(FITC-dextran)cardiac perfusion,and observed the retinal-choroidal structure changes of rats by Hematoxylin-eosin staining(HE)staining.We also detected the expression of Ang1,Rap1,GAPRap1,and vascular endothelial-cadherin(VE-cadherin)in the retinal-choroidal-sclera complex by Western blotting.Next,we cultured the rat choroidal vascular endothelial cells(RCVECs).When the cells were in the logarithmic growth phase,we stimulated these cells with vascular endothelial growth factor(VEGF)and cultured them for 24 hours,and divided into negative control group(siRNA-NC group),GAPRap1-siRNA group and GAPRap1-siRNA+Ang1 group.We further transfected cells with siRNA-NC(siRNA-NC group)or GAPRap1 small interfering RNA(GAPRap1-siRNA group and GAPRap1-siRNA+Ang1 group).In the GAPGAPRap1 small interfering RNA transfected cells,6 hours after transfection,we set aside some cells coculture with 200μg/L Ang1(GAPRap1-siRNA+Ang1 group).After another 24 hours,we extracted and quantified the expression levels of GAPRap1,Rap1,and VE-cadherin by Western blotting.We detected the expression of VE-cadherin using immunofluorescence.Results Compared with normal group,in model group,the neovascular leakage area and choroidal damage degree significantly increased(P<0.01),the expression of GAPRap1 and VE-cadherin proteins significantly reduced(P<0.05),and the expression of Rap1 had no significant change(P>0.05).Compared with model group,in the Ang1 treatment group the neovascular leakage area and choroidal damage degree were significantly reduced(P<0.01),the expression of GAPRap1 and VE-cadherin proteins in the choroid and cells significantly increased(P<0.01),the expression of Rap1 had no statistical change(P>0.05).In choroidal tissue,the expression of Ang1 protein in Ang1 treatment group was significantly higher than that in the other two groups(P<0.01).In the cell experiment,the expressions of GAPRap1 and VE-cadherin in GAPRap1-siRNA group were significantly lower than those in the GAPRap1-siRNA+Ang1 group and the siRNA-NC group(P<0.01),while the expression of Rap1 had no significant change(P>0.05).The immunofluorescence results showed that the fluorescence of VE-cadherin in the GAPRap1-siRNA group was significantly lower than that in the GAPRap1-siRNA+Ang1 group and siRNA-NC group(P<0.001).Conclusion Ang1 can reduce the leakage of choroidal neovascularization in rats,which has an inhibitory effect on CNV growth,and its mechanism may be related to the enhancement of cell adhesion through the GAPRap1-VE-cadherin pathway.