荧光抗体标记法在小鼠脉络膜新生血管中的应用  

Fluorescent antibody labeling for experimental choroidal neovascularization in mice

作　　者：顾丽萍[1] 陈莉[2] 陈辉[1] 庹旌生[1] 高小伟[1] 

机构地区：[1]南通大学附属医院眼科,226001 [2]南通大学病理教研室,226001

出　　处：《中华实验眼科杂志》2011年第7期619-624,共6页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景脉络膜新生血管（CNV）是导致多种眼底疾病视力损害的主要原因，准确建立CNV模型对于其实验和临床研究具有重要的意义，但目前尚无一种可重复且可靠的评估CNV模型及其有效治疗的方法。目的探讨荧光抗体标记法在定性和定量评价氪激光诱导的小鼠CNV中的应用价值。方法选取15只雄性SPF级C57BL／6J小鼠，用氪激光（波长为647．1am）视网膜光凝的方式建立CNV模型。分别于光凝后5rain，4、7、14及28d随机选取3只模型小鼠行眼底照相和荧光素眼底血管造影（FFA）检查。摘除眼球行脉络膜铺片，用荧光抗体（DAPI、isolectin-B4及phalloidin）分别标记光凝区域的细胞核、内皮细胞和肌动蛋白。用Imageproplus6．0软件测量CNV面积。结果FFA和脉络膜铺片检查结果显示激光光凝后5min及4d无CNV形成，光凝后7d开始出现CNV。光凝后7、14、28d有荧光素渗漏的光凝斑的百分率分别为76．47％（26／34）、81．81％（18／22）和50．00％（5／10）。脉络膜铺片后荧光显微镜检测结果显示，在正常的未光凝区域，视网膜色素上皮（RPE）细胞呈均匀一致的六边形排列；光凝后5min，在光凝部位可检测到一个环形荧光素缺损区，表明脉络膜-Bruch膜-RPE复合体已被破坏；光凝后4d出现一些细胞碎片以及核碎片，Bruch膜破损处可见自发荧光环。光凝后7d时，激光损伤区出现界限清楚的CNV网，并持续到28d。7、14及28d时根据脉络膜铺片所测得的CNV面积分别为（7．99±0．42）×10^3、（16．89±8．77）×10^3、（14．37±4．02）×10^3μm^2，差异有统计学意义（F=17．340，P=0．000）。光凝后14d和28d脉络膜铺片上CNV面积相差不大，但与7d时相比面积均明显增加（q=16．46、q=15．54，P〈0．01）。结论荧光抗体标记法不仅能很好地显示氪激光诱导的小鼠实验性CNV及其形态，而且能测定CNV的面积，为抗新生血管药�Background Choroidal neovascularization （CNV） is a main cause of visual impairment in many retinal diseases. To create an ideal CNV animal model is very important for the experimental and clinical study of CNV. The assessment method of repeatable and reliable for CNV model is still seldom. Objective This experiment was to explore the label value of fluorescent antibody for visualizing and quantifying the morphologie changes associated with laser-induced CNV. Methods Laser-induced CNV models were created in 30 eyes of 15 male SPF C57BL/6J mice by Krypton red laser irradiating fundus 2 spots around the optical disc with the wavelength 647. 1 nm, power 260 mW,spot diameter 50 μm and exposure time 0.05 seconds. The CNV was evaluated at 5 minutes,4,7,14 and 28 days after laser injury by using fundus photography and fundus fluorescein angiography （FFA）, and the successful models were identified as the rupture of Bruch＇ s membrane. The mice were then immediately sacrificed and the eyeballs were enucleated to prepare the choroidal flatmounts. The posterior eye cups were fluorescently labeledwith markers of cell nuclei （ DAPI,4＇ ,6＇ -diamino-2-phenylindole） , endothelial ceils （ isolectin-B4 ） , and filamentous actin （ phalloidin）. The CNV areas from specimens were measured by Image pro plus 6.0. Two eyes from one matched mouse without receiving photocoagulation were used as the coutrolls. This study followed the Standard of Association for Research in Vision and Ophthalmology. Results No any CNV was seen in photocoagulated eyes in 5 minutes and 4 days after laser irradiation. The first sign of CNV appeared at 7 days following photocoagulation. The incidence offluorescein leakage was 76.47% （26/34）, 81.81% （18/22）,50.00% （5/10） at 7, 14 and 28 days, respectively. The fluomicroscope examination showed that in unphotocoagulated areas, retinal pigment epithelial （RPE） cells were visualized wiib a uniform hexagonal array. Immediately after laser exposure, a circular area devoid of fl

关 键 词：脉络膜新生血管 激光光凝术 荧光抗体 

分 类 号：R773.4[医药卫生—眼科]