摘要目的 观察玻璃体腔注射脂质体介导的过氧化物酶增殖激活受体-γ共激活子-1α(PGC-1α)特异性小干扰RNA(siRNA)对小鼠视网膜新生血管的抑制作用.方法 80只C57BL/6J小鼠分为正常组、模型空白组、模型对照组和PGC-1α siRNA组,每组20只.正常组小鼠在正常空气中饲养.模型空白组、模型对照组和PGC-1α siRNA组小鼠建立氧诱导视网膜病变模型.小鼠12日龄时,模型对照组、PGC-1α siRNA组小鼠玻璃体腔分别注射阴性对照siRNA-脂质体复合物、PGC-1α siRNA-脂质体复合物1 μl;模型空白组小鼠不作处理.小鼠17日龄时,作视网膜铺片,荧光显微镜观察各组小鼠视网膜血管形态变化;作视网膜切片,光学显微镜下计数各组突破视网膜内界膜的新生血管内皮细胞核数;实时聚合酶链反应(PCR)检测各组小鼠视网膜中PGC-1α、血管内皮生长因子(VEGF)的mRNA表达;蛋白免疫印迹法(Western blot)检测各组小鼠视网膜中PGC-1α、VEGF的蛋白表达.计算PGC-1α siRNA组PGC-1α、VEGF mRNA和蛋白表达的抑制效率.结果 荧光显微镜观察发现,正常组小鼠视网膜血管分布呈网状;模型空白组、模型对照组小鼠视网膜可见大片无灌注区及新生血管丛,伴荧光渗漏;PGC-1α siRNA组小鼠视网膜无灌注区及新生血管丛较模型空白组、模型对照组明显减少.光学显微镜观察发现,模型空白组、模型对照组突破视网膜内界膜的血管内皮细胞核数较正常组明显增多,差异有统计学意义(P＜0.05);PGC-1α siRNA组突破视网膜内界膜的血管内皮细胞数较模型空白组明显下降,差异有统计学意义(P＜0.05).实时PCR和Western blot检测结果显示,模型空白组、模型对照组小鼠视网膜中PGC-1α、VEGFmRNA和蛋白表达均较正常组明显上调,差异有统计学意义(P＜0.05);PGC-1α siRNA组小鼠视网膜中PGC-1α、VEGF mRNA和蛋白表达较模型空白组明显下调,差异有统计学意义(P＜0.05).PGC-1αsiRNA组PGC-1α、VEGF mRNA表达的抑制效率分别为54％、48％,蛋白表达的抑制效率分别为53％、40％.结论 玻璃体腔注射脂质体介导的PGC-1α siRNA能有效抑制小鼠视网膜新生血管的形成.

abstractsObjective To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α) on retinal neovascularization in the mouse.Methods Eighty seven-day-old C57BL/6J mice were divided into normal group,model blank group,model control group and PGC-1α siRNA group,twenty mice in each group.Mice in the normal group were kept in normal room air.Mice in the model blank group,model control group and PGC-1α siRNA group were induced for retinal neovascularization by hypoxia.Liposome with PGC-1α siRNA (1 μl) and liposome with negative control siRNA (1 μl) were injected into the vitreous in the PGC-1α siRNA group and model control group respectively when mice were moved out to room air from the cabin (Postnatal 12).No injection were performed in the model blank group.At postnatal 17,fluorescein angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections.PGC-1α and vascular endothelial growth factor (VEGF) level in retina were measured by real-time polymerase chain reaction (real-time PCR) and Western blot.Inhibition efficiency of PGC-1α siRNA on PGC-1α and VEGF was calculated.Results Mice in the normal group showed reticular distribution of retinal blood vessels.Central nonperfused retina,neovascular tufts and fluorescein leakage were seen in the model blank group and model control group.Neovascular tuft and fluorescein leakage were decreased in the PGC-1α siRNA group compared to the model blank group and model control group.The neovascular nuclei were increased in the model blank group and model control group compared to the normal group (P＜0.05).The neovascular nuclei were decreased in the PGC-1α siRNA group compared to the model blank group and model control group (P＜0.05).The expression of PGC-1α mRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group,while decreased 54％ and 53％ respectively in the PGC-1α siRNA group as compared with model blank group and model control group (P＜0.05).The expression of VEGF mRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group,while decreased significantly in the PGC-1α siRNA group (decreased 48 ％ and 40 ％ respectively) as compared with model blank group and model control group (P＜0.05).Conclusions Intravitreal injection of PGC-1α siRNA mediated by liposome can inhibit retinal neovascularization in the mouse effectively.