γ-分泌酶抑制剂对视网膜新生血管形成的抑制作用及Nocth1信号通路调控机制  

Inhibitory effect of gamma-secretase inhibitor on retinal neovascularization and regulative mechanism of Notch1 signal pathway

作　　者：程志兴 谢洁 孟倩丽[1] Cheng Zhixing;Xie Jie;Meng Qianli(Department of Ophthalmology,Guangdong Provincial People's Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,China)

机构地区：[1]广东省人民医院眼科、广东省医学科学院,广州510080

出　　处：《中华实验眼科杂志》2022年第11期1031-1037,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(82171072);广东省自然科学基金项目(2021A1515010921)。

摘　　要：目的研究Notch1信号通路抑制剂γ-分泌酶抑制剂(DAPT)对氧诱导视网膜病变(OIR)小鼠视网膜新生血管形成的抑制作用及其可能机制。方法将SPF级出生后第7天(P7)的幼鼠与C57BL/6J母鼠共同饲养于含75%氧气的氧箱中5 d,然后放回正常氧气环境下饲养,以制备OIR模型。采用随机数表法将54只OIR小鼠分为OIR组、OIR+DMSO组和OIR+DAPT组,每组18只,所有小鼠右眼作为实验眼。OIR+DAPT组和OIR+DMSO组在P14小鼠玻璃体腔内分别注射10 mmol/L DAPT和DMSO溶液1μl,OIR组不予注射。P17时处死各组小鼠,摘出眼球后分离视网膜,采用Western blot法测定Notch1信号通路蛋白及其下游Hes1蛋白、视网膜小胶质细胞M1型标志物诱导型一氧化氮合酶(iNOS)和M2型标志物精氨酸酶-1(Arg-1)蛋白的相对表达量;制备视网膜铺片,采用同工凝集素(IB4)染色视网膜血管,计算小鼠视网膜新生血管相对面积,定义为新生血管面积/视网膜总面积×100%;采用苏木素-伊红染色法观察突破视网膜内界膜的血管内皮细胞数量。结果OIR+DAPT组、OIR组和OIR+DMSO组小鼠视网膜中Notch1蛋白相对表达量分别为0.68±0.06、1.00±0.00和1.03±0.08,Hes1蛋白相对表达量分别为0.70±0.08、1.00±0.00和1.02±0.07,组间总体比较差异有统计学意义(F=70.62、53.65,均P<0.01)。OIR+DAPT组小鼠视网膜中Notch1和Hes1蛋白相对表达量均明显低于OIR组和OIR+DMSO组,差异均有统计学意义(均P<0.01)。OIR+DAPT组、OIR组和OIR+DMSO组小鼠视网膜中Arg-1蛋白相对表达量分别为1.49±0.12、1.00±0.00和0.94±0.07,iNOS蛋白相对表达量分别为0.74±0.07、1.00±0.00和1.04±0.10,组间总体比较差异均有统计学意义(F=89.32、31.63,均P<0.01),与OIR组和OIR+DMSO组比较,OIR+DAPT组小鼠视网膜中Arg-1蛋白相对表达量明显升高,iNOS蛋白相对表达量明显下降,差异均有统计学意义(均P<0.01)。OIR+DAPT组、OIR组和OIR+DMSO组小鼠视网膜新生血管相对面积分别为(8.82Objective To investigate the inhibitory effect and underlying mechanism of gamma-secretase inhibitor blocking Notch1 signaling on retinal neovascularization caused by oxygen-induced retinopathy(OIR)in mice.Methods To establish the OIR model,7-day-old pups of C57BL/6J mice were exposed to 75%oxygen together with their mother until postnatal day(P)12.On P12,the mice were transferred to room air.All the mice were randomly divided into three groups,OIR group as control group,OIR+DAPT group and OIR+DMSO group receiving 1μl intravitreal injection of gamma-secretase inhibitor(DAPT,10 mmol/L)and 1∶20 DMSO dilution respectively.The right eye was taken as experimental eye.The mice were euthanized on P17 and the eyes were harvested to obtain retinas for further investigation.The total proteins were extracted from the retinas.The relative expression levels of Notch1 signal pathway and its downstream Hes1,the markers of M1 phenotype inducible nitric oxide synthase(iNOS)and M2 phenotype arginase-1(Arg-1)microglia were measured by western blot.Retinal flat mounts were made and the retinal vessels were stained with isolectin B4(IB4)to investigate the relative retinal neovascularization areas which was calculated as the ratio of neovascularization area/retinal area.The mumber of the neovascular endothelium cells beyond the inner limiting membrane was observed by hematoxylin-eosin staining.The use and care of animals complied with ARVO statement.This study protocol was approved by the Animal Ethics Committee of Guangdong Provincial People's Hospital(No.KY-Z-2021-2015-01).Results The relative protein expression levels of Notch1 and Hes1 in OIR+DAPT group,OIR group,and OIR+DMSO group were 0.68±0.06 and 0.70±0.08,1.00±0.00 and 1.00±0.00,1.03±0.08 and 1.02±0.07,respectively,with statistically significant differences among them(F=70.62,53.65;both at P<0.01).Compared with the OIR group and OIR+DMSO group,the expressions of Notch1 and Hes1 were significantly reduced in OIR+DAPT group(all at P<0.01).The relative protein expressio

关 键 词：小胶质细胞 视网膜新生血管 Γ-分泌酶抑制剂 Notch1信号通路 氧诱导视网膜病变 小鼠 

分 类 号：R774.1[医药卫生—眼科]