Ras相关的C3肉毒毒素底物1的小发夹RNA干扰活性氧-核因子κB通路抑制小鼠视网膜新生血管生成

Ras-related C23 botulinum toxin substrate 1 small hairphin RNA suppress mouse retinal neovascularization in mice

目的 观察Ras相关的C3肉毒毒素底物1的小发夹RNA（Racl-shRNA）在小鼠氧致视网膜病变中对视网膜新生血管（RNV）生成的抑制作用及对活性氧（ROS）-核因子κB（NF-κB）通路的影响.方法 将108只7日龄C57BL/6J小鼠随机平均分为3组.其中2组Smith法建立氧致视网膜病变模型;11日龄时玻璃体腔注射Racl-shRNA表达质粒或无意义质粒,分别作为基因干预组和空白对照组.第3组于正常氧环境中饲养,11日龄时玻璃体腔注射Racl-shRNA表达质粒作为空白干预组.15、17日龄时行荧光视网膜铺片,观察视网膜血管发育及新生血管情况.17日龄时计数眼球切片中突破内界膜的血管内皮细胞核数.17日龄时进行原位杂交法和荧光实时定量逆转录-聚合酶链反应法检测小鼠视网膜Racl和NF-κB p65亚单位的mRNA含量;免疫组织化学和蛋白质免疫印记检测Racl和NF-κB p65的蛋白表达.结果 基因干预组视网膜Racl的mRNA水平较空白对照组明显下调（t=4.500,P=0.001）.与空白对照组比较,基因干预组视网膜铺片中无灌注区、荧光渗漏和新生血管丛明显减轻,突破内界膜的血管内皮细胞核显著减少（t=6.521,P〈0.001）;视网膜NF-κB p65的核易位水平明显下降（t=16.008,P〈0.001）,同时mRNA表达亦明显下调（t=3.354,P=0.006）,与Racl mRNA表达呈正相关（r=0.580,P=0.012）.结论 小鼠玻璃体腔注射脂质体包裹的Racl-shRNA表达质粒可有效沉默视网膜Racl基因表达,阻断ROSNF-κB通路而参与抑制相对缺氧状态下RNV生成.

Objective To investigate the effects of knocking down Racl gene （ras-related C3 botulinum toxin substrate 1） by small hairpin RNA （shRNA） on retinal neovascularization in a mouse model of oxygen-induced retinopathy （OIR）. Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly. The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Racl-shRNA plasmid or the nonsense plasmid in the gene-intervention group and control group respectively at the age of postnatal day 11 （P11）. Non-OIR mice also received an intravitreal injection of Racl-shRNA plasmid at P11 as the blank-intervention group which lived in the normoxic environment. Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17. The expression of Racl and NF-κB p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction （RT-PCR） and in situ hybridization. Results Compared with the blank-control group, the level of Racl mRNA in the geneintervention group decreased obviously（t=4.5, P = 0. 001 ）; the retinal non-perfusion areas, fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t = 6. 521, P〈 0. 001） ; the level of NF-κB p65 nuclear translocation decreased（t= 16. 008, P〈0. 001）while the expression of NF-κB p65 mRNA was reduced obviously（t=3. 354, P=0. 006）, which was positively correlated with the expression of Ratl mRNA （P=0. 012）.Conclusion Intravitreal injection of Racl-shRNA with liposome in mice can effectively inhibit the expression of Racl, and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-κB pathway.