Radixin shRNA对高氧诱导的小鼠视网膜新生血管形成的抑制作用

Inhibitory effect of radixin shRNA on retinal neovascularization induced by hyperoxia in mice

背景视网膜新生血管性疾病是多种眼科疾病的病理基础，迄今为止新生血管形成的发病机制尚不完全清楚。研究显示，视网膜新生血管形成过程中radixin表达量明显增加，因此推测在视网膜新生血管相关疾病中抑制或沉默radixin基因有望成为治疗的新方法。目的研究radixin小发卡（shRNA）干扰质粒对氧诱导视网膜病变（OIR）小鼠模型视网膜中radixin基因表达的抑制作用，观察其对小鼠视网膜新生管形成的影响。方法将64只出生后7d的SPF级C57BL／6J小鼠按照随机数字表法分为正常对照组、模型对照组、radixin shRNA质粒组和shRNA质粒组，其中模型对照组、radixin shRNA质粒组和shRNA质粒组小鼠在体积分数（75±2）％的氧环境中饲养5d，建立OIR动物模型，正常对照组小鼠饲养于正常氧环境下。radixin shRNA质粒组和shRNA质粒组小鼠于出生后第12天分别于玻璃体腔注射1μg radixin shRNA质粒和对照shRNA质粒，小鼠出生后第17天，对各组小鼠行FD-2000S血管造影术，制备视网膜铺片，观察视网膜血管形态和分布；摘取各组小鼠眼球制备视网膜切片，采用视网膜苏木精－伊红染色法观察突破视网膜内界膜的血管内皮细胞核和新生血管；应用免疫组织化学染色法检测radixin在视网膜中的表达分布；分别采用实时荧光定量PCR法和Western blot法检测radixin mRNA及其蛋白在视网膜组织中的表达情况。结果正常对照组小鼠视网膜铺片显示视网膜血管走行正常，模型对照组小鼠视网膜后极部大片状无灌注区，可见大血管迂曲和血管壁荧光素渗漏和新生血管，shRNA质粒组小鼠视网膜可见无灌注区和微血管瘤，而radixin shRNA质粒组小鼠视网膜后极部无灌注区面积较小，血管迂曲和渗漏现象较模型对照组和shRNA质粒组明显减轻。视网膜组织病理学检查显示，模型对照组和shRNA质粒组小鼠视网膜内界膜不连续，可见大量血管内皮细胞核和血管管腔突破内界膜，radixin shRNA质粒组视网膜内界膜形态接近正常对照组，有少量血管内皮细胞核和血管管腔突破内界膜。免疫组织化学检查显示，正常对照组和radixin shRNA质粒组radixin表达弱于模型对照组和shRNA质粒组。正常对照组、模型对照组、radixin shRNA质粒组和shRNA质粒组小鼠视网膜中radixinmRNA的相对表达量分别为1．002±0．043，2．236±0．093，0．556±0.015和2．272±0．096，各组间总体比较差异有统计学意义（F=504．545，P＝0．000）。正常对照组、模型对照组、radixin shRNA质粒组和shRNA质粒组小鼠视网膜中radixin蛋白的相对表达量分别为1．000±0．082、1．193±0．021、0．263±0．016和1．235±0．005，各组间总体比较差异有统计学意义（F=753．522，P=0．000）radixin shRNA质粒组radixin mRNA和蛋白的相对表达量较模型对照组和shRNA质粒组明显减低，差异均有统计学意义（均P〈0．01）。结论Radixin shRNA能有效沉默OIR动物模型视网膜中radixin基因的表达，抑制视网膜新生血管的形成。

Background Retinal neovascularization is pathological basis of a variety of fundus diseases,but its pathogenesis is unclear. Studies showed that the expression level of radixin in retina is remarkably increased in retinal neovascularization-related diseases. It is presumed that silencing or down-regulating the abnormal expression of radixin is helpful for curing retinal neovascularization-related diseases. Objective This study was to investigate the inhibitory effect of radixin short hairpin RNA （shRNA） plasmid on expression of radixin gene in retina of oxygeninduced retinopathy （OIR） mice. Methods Sixty-four 7-day-old C57BL/6J mice were randomly divided into normal control group, model control group, radixin shRNA plasmid group and shRNA plasmid group by random number table. There were 16 mice in every group. OIR models were established by exposing the mice in an environment of （75±2） % oxygen for 5 days and then returned to the normal air in the model control group, radixin shRNA plasmid group and shRNA plasmid group,while the mice of the normal control group were fed in the normal air environment. Radixin shRNA plasmid or control shRNA plasmid at the dose of 1 μg was intravitreally injected in 12-day-old mice of the radixin shRNA plasmid group or shRNA plasmid group,respectively. Five days later, FD-2000S angiography was performed on the mice of each group and then retinal flatmounts were prepared for the observation of retinal vessels. The mice from various groups were sacrificed and retinal sections were prepared. The vascular endothelial nucleus and new blood vessels extending inner limiting membrane （ILM） were examined by hematoxylin and eosin staining;the expression of radixin in the retinas was detected using immunochemistry; the relative expression levels of radixin mRNA and protein were quantitative assayed by real-time quantitative RCR and Western blot, respectively. The use and care of the animals adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Results The distribution of retinal vessels was normal in the normal control group. Non-perfusion zone at the posterior pole of retina, circuity of blood vessels,leakage of vessel wall and new blood vessels were found in the mice of the model control group. Non-perfusion zone and microaneurysms were also exhibited in the shRNA plasmid group. However,these findings were slight in the radixin shRNA plasmid group. The surface of ILM was in discontinuity in the model mice and shRNA-injected mice with more vascular endothelial cell nucleus and more tubes extending ILM than that in the radixin shRNA plasmid group. The immunochemistry results showed that the expressions of radixin in the normal control group and radixin shRNA plasmid group were weaker than those in the model control group and control shRNA plasmid group. The relative expression levels of radixin mRNA were 1. 002±0. 043,2. 236±0. 093,0. 556±0. 015 and 2. 272±0. 096 in the normal control group, model control group,radixin shRNA plasmid group and control shRNA plasmid group,and those in the radixin shRNA plasmid group were significantly reduced in comparison with the normal control group, model control group and the shRNA plasmid group （all at P〈0. 01 ）. The relative expression levels were 1. 000±0. 082,1. 193±0. 021,0. 263±0. 016 and 1. 235±0. 005 in the normal control group,model control group,radixin shRNA plasmid group and shRNA plasmid,with the lowest expression level in the radixin shRNA plasmid group （ all at P〈0. 01 ）. Conclusions Radixin shRNA can downregulate the expression of radixin gene in the retinas of OIR mice and further inhibit pathological retinal neovascularization.