15-脂氧合酶-1基因转移抑制小鼠视网膜新生血管的实验研究

摘要

目的 观察15-脂氧合酶-1(15-LOX-1)基因转移对氧诱导小鼠视网膜新生血管的抑制作用.方法 7日龄C57BL/6J小鼠96只,随机分为正常对照组、氧诱导视网膜病变(OIR)模型组、基因治疗组和空白载体组.将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.小鼠出生后第12天基因治疗组玻璃体腔注射携带增强型绿色荧光蛋白(EGFP)和小鼠15-LOX-1基因的重组腺病毒(Ad-15-LOX-1-EGFP)载体1μl；空白载体组注射等量携带EGFP的重组腺病毒(Ad-EGFP)载体.注射后第2天行视网膜铺片荧光显微镜观察EGFP的表达.注射后第5天行免疫荧光染色法、实时荧光定量聚合酶链反应和蛋白免疫印迹法检测15-LOX-1基因转染视网膜的表达；视网膜铺片观察视网膜血管变化,测量视网膜无灌注区和新生血管的相对面积；石蜡切片苏木精-伊红染色并计数突破视网膜内界膜的血管内皮细胞核.结果 Ad-15-LOX-1-EGFP注射第2天,视网膜铺片上观察到EGFP的表达.免疫荧光染色结果显示,15-LOX-1基因转染视网膜主要表达在外丛状层、内核层和神经节细胞层.基因治疗组15-LOX-1蛋白和mRNA表达水平明显高于OIR模型组和空白载体组,差异有统计学意义(t蛋白表达水平=22.74、24.13,tmRNA表达水平=12.51、13.40;P＜0.01)；基因治疗组视网膜无灌注区和新生血管面积较OIR模型组和空白载体组显著减小,差异有统计学意义(t血管区面积=16.22、14.31,t新生血管面积=9.97、9.07;P＜0.01)；基因治疗组中突破视网膜内界膜的血管内皮细胞核与OIR模型组和空白载体组比较明显减少,差异有统计学意义(t=14.25、11.62,P＜0.01).结论 15-LOX-1基因转移不仅可以减少氧诱导小鼠视网膜无灌注区面积,并且对视网膜新生血管有显著的抑制作用.%Objective To investigate the inhibitory effects of 15-lipoxygenase-1 (15-LOX-1) gene transfer on oxygen-induced retinal neovascularization in mice.Methods Ninety-six 7-day-old C57BL/6J mice were randomly divided into normal control group,oxygen-induced retinopathy (OIR) model group,gene treated group and empty vector group.The mice with their mothers were kept in (75 ± 2) ％ 02environment for 5 days and then returned to normoxia for 5 days to establish the OIR model.At postnatal day 12,the gene treated group received intravitreous injection of recombinant adenovirus (Ad) vector containing both enhanced green fluorescent protein (EGFP) and mouse 15-LOX-1 genes (Ad-15-LOX-1-EGFP) at 1 l,while the empty vector group received the same volume of recombinant Ad vector containing EGFP (Ad-EGFP).The expression of EGFP was observed on flat-mounted retina by fluorescence microscopy 2 days after intravitreous injection of Ad-15-LOX-1-EGFP.At postnatal day 17,the efficacy of 15-LOX-1 gene transfer on retinal tissue was detected by immunofluorescence staining,real-time polymerase chain reaction and Western blot.The changes of retinal vessels,relative retinal non-perfusion and neovascularization areas were evaluated by fluorescein isothiocyanate-dextran fluorescein angiography on flatmounted retina.The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section.Results Two days after intravitreous injection of Ad-15-LOX-1-EGFP,the expression of EGFP had been seen by fluorescence microscopy on flat-mounted retina.Immunofluorescence staining of retinal section revealed that 15-LOX-1 expression was primarily in the outer plexiform layer,inner nuclear layer and ganglion cell layer of retina.The 15-LOX-1protein and mRNA expression levels were higher in gene treated group than those in OIR model group and empty vector group (tprotein =22.74 and 24.13 respectively,tmRNA =12.51 and 13.40 respectively; P＜0.01).The relative retinal non-perfusion and neovascularization areas were significantly smaller in gene treated group than those in OIR model group and empty vector group (tnon-perfusion =16.22 and 14.31respectively,tnerovascularization =9.97 and 9.07 respectively; P＜0.01),and the number of endothelium cell nuclei breaking through the ILM in gene treated group was obviously lower than the other two groups (t=14.25 and 11.62 respectively,P＜0.01).Conclusion 15-LOX-1 gene transfer can decrease the oxygeninduced retinal non-perfusion areas and inhibit the retinal neovascularization in mice.