色素上皮衍生因子对氧诱导视网膜新生血管的抑制作用

Inhibitory effects of pigment epithelium derived factor on oxygen-induced retinal neovascularization

目的 观察色素上皮衍生因子（PEDF）对氧诱导视网膜新生血管（OIR）的抑制作用,初步探讨PEDF抑制新生血管形成的可能机制.方法 7日龄C57BL/6J新生小鼠140只,随机数字表法分为正常对照组、OIR模型组、PEDF治疗组、磷酸盐缓冲液（PBS）干预对照组.除正常对照组外,其余各组小鼠与哺乳母鼠共同置于氧浓度为（75±2）％的氧箱内饲养5d后转移至正常环境中饲养5d,建立OIR模型;正常对照组小鼠始终置于正常氧环境饲养;OIR模型组小鼠出氧箱后不作任何处理.12、14日龄时,PEDF治疗组小鼠玻璃体注射分别注射2 μg/A的PEDF 1 μl;PBS干预对照组小鼠玻璃体腔注射等量PBS.视网膜铺片、冰冻切片荧光染色观察视网膜新生血管生长情况;蛋白免疫印迹法（Western blot）观察小鼠视网膜白细胞介素（IL）-1β蛋白表达量;实时逆转录-聚合酶链反应（RT-PCT）检测小鼠视网膜IL-1β mRNA相对表达量.结果 视网膜铺片检查结果显示,OIR模型组视网膜新生血管面积较正常对照组明显增大,差异有统计学意义（t=15.02,P＜0.01）;PEDF治疗组视网膜新生血管面积较PBS干预对照组明显减小,差异有统计学意义（t=5.96,P＜0.01）.荧光显微镜观察结果显示,OIR模型组视网膜可见外丛状层（OPL）与神经节细胞层（GCL）之间的新生血管簇较正常对照组明显增多;PEDF治疗组视网膜OPL与GCL之间的新生血管簇较PBS干预对照组明显减少.Western blot检测结果显示,OIR模型组视网膜IL-1β蛋白表达水平较正常对照组显著提高,差异均有统计学意义（t=3.35,P＜0.05）.PEDF治疗组与PBS干预对照组比较,IL-1β蛋白表达水平下降,差异有统计学意义（P＜0.05）.正常对照组与PEDF治疗组比较,IL-1β蛋白表达水平无显著差异（F=11.764,P＞0.05）.实时RT-PCR检测结果显示,OIR模型组视网膜IL-1βmRNA相对表达量较正常对照组明显增加,差异有统计学意义（t=4.43,P＜0.01）.PEDF治疗组与PBS干预对照组比较,前者IL-1β mRNA相对表达量显著减少,差异有统计学意义;正常对照组与PEDF治疗组比较,IL-1β mRNA相对表达量无显著差异（F=11.151,P＞0.05）.结论 PEDF可抑制OIR视网膜新生血管的形成.下调IL-1β在视网膜的表达可能是其机制之一.

Objective To study the inhibitory effects of pigment epithelium derived factor （PEDF） on oxygen-induced retinal neovascularization in mice,and to investigate the possible involvement of interleukin-1β （IL-1β） in the neovascular-inhibitory function of PEDF.Methods A total of 140 postnatal day （P）7 C57BL/6 mice were randomly divided into normal control group,oxygen-induced retinopathy （OIR） model group,PEDF treatment group and PBS treatment control group.All mice except normal control group with their mothers were exposed to （75 ± 2）％ oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model.Mice in normal control group were kept in room air only.At P12 and P14,respectively,mice in PEDF treatment group received intravitreous injections of 1 μl PEDF （2 μg/μl）,while PBS treatment control group received the same volume of PBS （10 mmol/L,pH7.4）.All mice were euthanized at P17 and eyes were isolated.The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy.Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction （Real-time RT-PCR）.Results Changes of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina,the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group （t =15.02,P＜0.01）,and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group （t=5.96,P＜0.01）.Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer （OPL） to ganglion cell layer （GCL） of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group.The specific expression levels of IL-1β protein in retinas of OIR mice by Western blot analysis were higher than those of normal control group（t=3.35,P＜0.05）,While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group （P＜0.01）,and there were no difference in normal control group and PEDF-treated group （F=11.764,P＞0.05）.Similarly,expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group（t =4.43,P ＜ 0.01）.After treated with PEDF,expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group （P ＜ 0.01）,and there were no difference in normal control group and PEDF-treated group （F=11.15,P＞0.05）.Conclusions PEDF can inhibit oxygen-induced retinal neovascularization.The mechanism may be related to that PEDF can downregulate the expression of IL 1β in retina.