Toll样受体4对氧诱导视网膜新生血管发生的促进作用及其机制  

Promoting effect of Toll like receptor 4 on oxygen-induced retinal neovascularization and its mechanism

作　　者：徐文芹[1] 尹婕[1] 王雨生[1] 

机构地区：[1]第四军医大学西京医院眼科全军眼科研究所,西安710032

出　　处：《中华实验眼科杂志》2016年第4期317-323,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81271014、81470655、81570856）

摘　　要：背景研究发现，Toll样受体（TLRs）在视网膜新生血管的形成中发挥重要作用，但其作用机制尚不清楚。目的探讨TLR4对小鼠氧诱导视网膜病变（OIR）中新生血管生成的作用及其机制。方法将18只7日龄新生C57BL／6J小鼠以随机数字表法按小鼠窝别分为常氧组、OIR组及0IR＋脂多糖（LPS）-EB组，常氧组小鼠在正常氧环境中饲养，OIR组及OIR＋LPS—EB组小鼠于生后第7天（P7）与母鼠置于体积分数（75±2）％高氧氧箱中饲养5d以诱导OIR模型，于P12在正常氧环境中饲养，OIR＋LPS-EB组P12小鼠腹腔内注射LPS-EB，剂量为1mg／kg，OIR组同法注射PBS。各组摘取P17小鼠眼球于质量分数4％多聚甲醛中固定并行视网膜铺片和Lectin染色，计算视网膜无血管区和新生血管区面积占全视网膜的百分比。各组制备P17小鼠眼后节组织冰冻切片并行免疫荧光检测，计数小鼠5mm视网膜全长活化小胶质细胞数目；采用CDllb和TLR4免疫荧光双标记法检测各组小鼠视网膜小胶质细胞中CDllb、TLR4的表达及其分泌血管内皮生长因子（VEGF）、白细胞介素1p（IL-1β）和肿瘤坏死因子-a（TNF-a）的水平。结果常氧组P17小鼠视网膜血管分布和形态正常，OIR组和OIR＋LPS—EB组P17小鼠视网膜中央均出现无血管区和新生血管簇，OIR＋LPS-EB组小鼠视网膜无血管区面积比为（18．47±1．32）％，新生血管面积比为（3．29±0．85）％，分别大于OIR组的（15．78±1．44）％和（1．77±0．19）％，差异均有统计学意义（t=3．36，P=0．01；t=4．22，P=0．00）。免疫荧光结果显示，常氧组P17小鼠视网膜中活化小胶质细胞数量极少，OIR组和OIR＋LPS—EB组P17小鼠视网膜中活化小胶质细胞荧光强度增强，活化小胶质细胞数量增加，其中OIR＋LPS-EB组小鼠视网膜中小胶质细胞数明显多于OIR组，差异有统计学意义[（95．50±4．77）／5mm与（74．83±4．Background Studies showed that Toll like receptors （TLRs） play an important role in the development of retinal angiogenesis. However, the effect and mechanism of TLR4 affecting angiogenesis in oxygen induced retinopathy （OIR） had not been clarified yet. Objective This study aimed to investigate the mechanism of TLR4 in oxygen induced retinal neovascularization in mice. Methods Eighteen 7-day-old （P7） C57BL/6J mice were randomly divided into normoxic group, OIR model group and OIR±lipopolysaccharide （LPS）-EB group. OIR models were established by exposing the mice with maternal mouse to 75% oxygen environment for 5 days, then 1 mg/kg LPS-EBO and PBS were intraperitoneally injected in P12 mice in the OIR±LPS-EB group and OIR modelgroup, respectively. Whole retinal flatmounts of P17 mice were prepared and Lectin staining was performed to calculate the ratio of avascular and neovascular area to retina area. The frozen sections of the posterior ocular segment were prepared, and the number of activated microglial cell was detected by immunofluorescence technique, and the expressions of CDllb and TLR4 in the microglial cells as well as the levels of vascular endothelial growth factor （VEGF） ,interleukin-lβ （IL-1β）, tumor necrosis factor-a （TNF-a） were assayed by fluorescence double labeling method. The use and care of the experimental animals complied with ARVO Statement. Results The distribution and morphology of retinal vessels were normal in P17 mice of the normoxic group, and avascular and new blood vessels cluster were found in the mice of the OIR group and OIR±LPS-EB group. The ratio of avascular area was （ 18.47± 1.32） % and that of the new blood vessel area was （ 3.29 ±0. 85 ） % in the OIR＋LPS-EB group, which were increased in comparison with （15.78±1.44）% and （1.77±0.19）% of the OIR group （t = 3.36,P=0.01;t=4.22, P = 0.00）. Immunofluorescence results displayed a few Ibal microglia cells in the normoxic group, the cells number were significantly higher i

关 键 词：TOLL样受体4 视网膜新生血管 血管生成 缺血/病理 神经胶质细胞/代谢 动物模型 近交系C57BL小鼠 炎性介质/代谢 信号转导 

分 类 号：R774.1[医药卫生—眼科]