MEK/ERK和PI3K/Akt通路对大鼠脉络膜新生血管中DDR2和MMP-13表达的调控作用  

Modulation of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in choroidal neovascularization

作　　者：杨秀梅[1] 王雨生[2] 张建[3] 李燕[3] 药立波[3] 

机构地区：[1]北京军区总医院眼科,100700 [2]第四军医大学西京医院眼科全军眼科研究所,西安710032 [3]第四军医大学细胞与分子生物学实验室,西安710032

出　　处：《中华实验眼科杂志》2015年第8期678-685,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81070748）;国家重点基础研究发展计划（973）项目（2011CB510200）;志谢感谢第四军医大学西京医院眼科全军眼科研究所马吉献技师对本实验提供的技术支持;同时,本研究得到德国洪堡基金会（Alexanderyon Humboldt Foundation）仪器设备捐赠基金（V-8151/02085,ToYSWang）资助,特此感谢

摘　　要：背景 研究表明盘状结构域受体2（DDR2）和基质金属蛋白酶-13（MMP-13）在肿瘤新生血管发生中具有重要作用,是相关疾病治疗的关键靶点,但DDR2和MMP-13在脉络膜新生血管（CNV）发生中的作用尚不清楚.目的 探讨DDR2和MMP-13在大鼠CNV中的表达特点以及MEK/ERK和PI3 K/Akt通路对DDR2及MMP-13表达的调控作用.方法 采用532 nm倍频激光视网膜光凝法制备棕色挪威（BN）大鼠CNV模型,将78只BN模型鼠按照随机数字表法分为正常对照组（n=7）、模型对照组（n=39）、PD98059（MEK1抑制剂）组（n=16）和LY294002（PI3K抑制剂）组（n=16）,PD98059组和LY294002组大鼠分别于光凝后1d、7d玻璃体内注射5 mmol/L PD98059 3μl或5 mmol/L LY294002 3μl.分别于光凝前和光凝后1、3、7、14 d用过量麻醉法处死动物并制备样本和切片,采用Western blot法检测各组大鼠眼杯中ERK、p-ERK、Akt和p-Akt蛋白的表达变化,采用逆转录PCR（RT-PCR）法检测各组大鼠眼杯中DDR2 mRNA和MMP-13 mRNA的表达变化,采用组织病理学方法测定各组大鼠CNV厚度,采用免疫组织化学和免疫荧光染色法分别定位和测定大鼠CNV区域DDR2和MMP-13的表达.结果 光凝后3d,光凝局部细胞增生,光凝后7 d CNV形成,至光凝后14 d CNV趋于稳定.免疫组织化学染色结果显示,DDR2在正常大鼠视网膜血管内皮细胞层、视网膜神经节细胞（RGCs）层和内核层细胞中呈弱表达,但在CNV中呈强阳性表达;MMP-13在正常对照组大鼠视网膜内界膜层、光感受器层和巩膜层均呈强阳性表达,在模型对照组中呈强阳性表达.免疫荧光结果显示,CNV组织中DDR2和MMP-13共表达.RT-PCR结果显示,模型对照组光凝后1、3、7、14 d DDR2 mRNA相对表达水平分别为55.22±4.03、47.74±2.23、14.82±4.56和5.59±2.47,MMP-13 mRNA相对表达水平分别为25.54±3.83、43.51±4.36、10.90±4.00和5.23±3.23,与正常对照组相比,差异均有统计学意义（均P＜0.05）.免疫�Background It is estimated that discoidin domain receptor 2 （DDR2） and matrix metalloproteinase-13 （MMP-13） play an important role in the development of tumor angiogenesis.However,their effects on choroidal neovascularizaiton （CNV） have not been clarified yet.Objective This study was to observe the expression pattern of DDR2 and MMP-13 in CNV and to further investigate the regulation role of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in CNV.Methods CNV models were established in 78 Brown Norway （BN） rats by retinal photocoagulation with 532 nm laser.Then the animals were randomly divided into the normal control group （n =7）,the model control group （n =39）,PD98059 （MEK1 inhibitor） group （n =16） and LY294002 （PI3K inhibitor） group （n =16）,and 5 mmol/L PD98059 or 5 mmol/L LY294002 3 μl was intravitreally injected 1 day and 7 days after photocoagulation in the PD98059 group or LY294002 group.The expression of DDR2 and MMP-13 mRNA and proteins in the CNV area were detected by using reverse transcription PCR （RT-PCR）,and the expression levels of p-ERK/ERK and p-Akt/Akt protein were detected by Western blot assay.CNV thickness was measured by pathological examination 14 days after photocoagulation,and the changes of CNV thickness,the expression levels of DDR2 and MMP-13 in CNV were compared among the model control group,PD98059 group and LY294002 group.Results Three days after photocoagulation,the cells within the lasered lesions proliferated,then CNV formed 7 days after photocoagulation and became stable 14 days after photocoagulation.Immunohistochemistry staining indicated that DDR2 was weakly expressed in the cells of ganglion cell layer,inner nuclear layer and vascular endothelial cells;while MMP-13 was strongly expressed in the cells of the inner limiting membrane layer,photoreceptor layer and sclera layer.Both DDR2 and MMP-13 were strongly expressed in CNV area.Double immunofluorescence staining revealed that MMP-13 and DDR2 co-expression in CNV

关 键 词：盘状结构域受体2 脉络膜/病理 新生血管 基质金属蛋白酶-13 疾病模型 BN大鼠 

分 类 号：R773.4[医药卫生—眼科]