氧诱导视网膜病变小鼠模型的蛋白表达谱分析  

Analysis of protein expression profiling in a mouse model of oxygen-induced retinopathy

作　　者：漆晨[1] 韩倩 薄其玉 刘勋 王飞 张琰 李筱荣[1] 

机构地区：[1]天津医科大学眼科医院 天津医科大学眼科研究所 天津医科大学眼视光学院,300384

出　　处：《中华实验眼科杂志》2015年第12期1069-1075,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：第45批留学回国人员科研启动基金;教育部博士点基金联合资助（20111202110008）;教育部博士点基金（20121202120005）

摘　　要：背景视网膜新生血管（RNV）是目前世界范围内主要的致盲原因之一，而目前的治疗效果并不理想，因此需要寻找在疾病状态下异常表达的蛋白质标志物，为新生血管性视网膜疾病的治疗提供新的靶点。目的观察氧诱导视网膜病变（OIR）小鼠模型中视网膜血管的形态学特征，筛选并验证差异性表达的蛋白质分子。方法选取7日龄健康清洁级C57BL／6J幼鼠44只，分为OIR组和正常对照组。OIR组幼鼠先在氧体积分数为（75±2）％的环境下饲养5d，然后在正常空气环境中饲养5d，而正常对照组幼鼠仅在正常空气环境下饲养10d。取17日龄幼鼠经球后静脉注射高分子量异硫氰酸葡聚糖（FITC-dextran），然后制备视网膜铺片，观察视网膜血管的形态并统计视网膜无灌注区相对面积；取各组小鼠眼球行石蜡切片和苏木精一伊红染色，计数突破内界膜的血管内皮细胞核数；采用蛋白芯片法检测蛋白质分子在OIR组和正常对照组小鼠眼球中的差异性表达，并用Western blot法和ELISA法对差异表达蛋白进行验证。结果视网膜铺片结果显示，OIR组小鼠视网膜周边血管紊乱，静脉血管迂曲，视网膜中央有明显的无灌注区，在视网膜灌注区和无灌注区的交界处有大量新生血管芽，而正常对照组视网膜血管细密，静脉平滑，分布均匀整齐。OIR组小鼠视网膜无灌注区的相对面积为（25．53±2．16）％，较正常组无灌注区的相对面积（0．66±0．36）％明显增加，差异有统计学意义（t=-27．61，P〈0．01）。石蜡切片苏木精-伊红染色结果表明，OIR组小鼠视网膜平均每张切片突人玻璃体腔的血管内皮细胞核数为（28．41±3．97）／切片，而正常组为（0．16±0．31）／切片，两组间比较差异有统计学意义（t=-54．42，P〈0．001）。蛋白芯片检测结果显示，在检测的62个与新生血管和炎症相关的细胞Background Retinal neovaseularization （RNV） is one of the major causes of blindness worldwide,but the pathogenic mechanism of this disease remains unclear, and therapeutic modalities need to be improved. Therefore, it is necessary to identify ocular protein markers with significant expression changes during RNV, thereby providing novel therapeutic targets for neovascular retinopathies. Objective This study was to investigate retinal vessel morphological characteristics and protein expression profiling in a mouse model of oxygen-induced retinopathy （OIR）. Methods Forty four C57BL/6J mouse pups were randomly divided into normal control group and OIR group at postnatal day 7 （PT）. The mice in the normal control group were raised under the normal air for 10 days. The mice of the OIR group were exposed to （75±2）% oxygen for 5 days. The mother mice were alternated between the two groups every day. The mice of the OIR group returned to normal air at P12. Fluorescein isothiocyanate-dextran （FITC-dextran） was retrobulbarly injected at P17 mice from both groups, and the retinal flatmounts were prepared after fixation. The FITC-dextran-labeled retinal vessels were observed and quantified; the paraffin sections of eyeballs were processed for hematoxylin and eosin staining,and the number of pre-retinal vascular cell nuclei was quantified. The total proteins were extracted from the eyes, and the expression profiling was analyzed by a customized protein array and verified by Western blot and ELISA. Results The retinal flatmounts labeled with FITC- dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds, and the vascular obliteration was prominent in the center of retina. Contrastly, the vessels were smooth,organized, and evenly distributed in the normal control group. The percentage of vascular obliteration area in the OIR group was （25.53±2. 16）% ,which was significantly higher than （0. 66±0. 36）% in the normal control group （t=

关 键 词：病理性新生血管/生理病理 氧诱导视网膜病变 蛋白标志物表达谱 靶点 近交系C57BL小鼠 

分 类 号：R774.1[医药卫生—眼科] R-332[医药卫生—临床医学]