摘要目的 探讨玻璃体腔注射靶向血管内皮生长因子(VEGF)的RNA干扰慢病毒抑制氧诱导视网膜病变(OIR)小鼠视网膜新生血管生成的作用及其机制.方法 实验研究.构建4对针对靶基因小鼠VEGF的siRNA干扰载体,筛选并进行慢病毒包装.60只C57BL/6J小鼠分成4组(每组15只):正常对照组,OIR模型组,OIR+空载体组,OIR+VEGF-RNA干扰组.OIR+空载体组和OIR+VEGF-RNA干扰慢病毒组的小鼠在生后第5天玻璃体腔注射相应的1μl的7.5×107空载体慢病毒和VEGF-RNA干扰慢病毒.后3组小鼠在生后第7天建立OIR模型.第17天时FITC-Dextran灌注视网膜铺片观察4组小鼠视网膜血管形态及面积变化,视网膜铺片免疫荧光染色检测紧密连接蛋白Claudin-5和Occludin的分布变化,Western blot检测VEGF、磷酸肌醇3激酶(PI3K)、酪氨酸蛋白激酶SRC、磷酸化细胞外信号调节激酶( p-ERK)蛋白表达量的变化.数据采用单因素方差分析进行比较.结果 FITC-Dextran灌注视网膜铺片显示正常组视网膜血管分布呈均匀网状；RNA干扰组新生血管面积(0.271 399 mm2)明显较OIR模型组(1.212 782 mm2)、空载体组(1.152 504 mm2)少(F=449.924,P＜0.01).OIR模型组和空载体组间差异无统计学意义,其余两两间差异均有统计学意义(P＜0.01).视网膜铺片免疫荧光染色显示紧密连接蛋白Claudin-5和Occludin在RNA干扰组中与正常组相似,呈均匀光滑线性分布,而在OIR模型组、空载体组的分布中断、不均匀,在新生血管团中可见团块状的强荧光；VEGF的RNA干扰组中VEGF、PI3K、酪氨酸蛋白激酶SRC和p-ERK的蛋白表达量较OIR模型和空载体组低.结论 玻璃体腔注射靶向VEGF的RNA干扰慢病毒能有效抑制OIR小鼠模型中VEGF及其下游通路蛋白的表达,从而抑制视网膜新生血管的形成,为临床上早产儿视网膜病变的防治提供了新思路和新途径.

abstractsObjective To investigate the inhibitory effect of vascular endothelial growth factor (VEGF)-targeted RNA interference on the retinal neovascularization of mice with oxygen-induced retinopathy (OIR).Methods This was an experimental study.OIR mice models were induced by exposing mice to a 75％ concentration of oxygen from postnatal day 7 (P7) to P12 and then returned to room air from P12 to P17.VEGF-siRNA was designed,screened and packaged in 293T cells to produce VEGF-RNAi-lentivirus.Sixty C57BL/6J mice were divided into 4 groups (every group n=15):normal control group,OIR group,OIR+lentivirus (OIR-K-virus) group,and OIR+VEGF-RNAi-lentivirus (OIR-Ri-virus) group.Intravitreal injection of lentivirus was performed at P5 in the OIR-K-virus group and OIR-Ri-virus group.After FITC-Dextran perfusion,a retinal flat-mount was used to observe the area of retinal neovascularization.The distribution of Claudin-5 and Occludin was detected by double immunofluorescence staining of the retinal flat-mount.The expression of VEGF,phosphotylinosital 3 kinase (PI3K),tyrosine kinase SRC,extracellular signal-regulated kinase (ERK) and phosphorylation ERK (p-ERK) proteins was analyzed by Western blot.Data were analyzed with a one-way ANOVA.Results A study of the retinal flat-mounts after FITC-Dextran perfusion showed that the distribution of retinal vessels was uniform and there was no neovascularization in the normal group.The area of retinal neovascularization in the OIR group was 1.212 782 mm2,OIR-K-virus group was 1.152 504 mm2,and OIR-Ri-virus group was 0.271 399 mm2,the difference between groups was significant (F=449.924,P＜0.01).There was no significant difference in the areas of retinal neovascularization between the OIR group and OIR-K-virus group.The areas of retinal neovascularization in the OIR-Ri-virus group were reduced significantly (P＜0.01) compared to the OIR group or the OIR-K-virus group.The distribution of Claudin-5 and Occludin in the retinal vessels of the OIR-Ri-virus group was more uniform and smoother than distribution in the OIR group or OIR-K-virus group.The expression of VEGF,PI3K,tyrosine kinase SRC and p-ERK protein in the OIR-Ri-virus group was significantly decreased compared to expression in the OIR and OIR-K-virus groups and was similar to the expression of the normal group.Conclusion The expression of VEGF and its signal pathway and development of retinal neovascularization in the OIR mice model were significantly inhibited by the intravitreal injection of lentivirus-mediated VEGF RNA interference,which may provide a new approach for the treatment of retinopathy of prematurity (ROP) in the clinic.