CAJ | 학술논문

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.
목적 관찰Ras상관적C3육독독소저물1적소발협RNA(Racl-shRNA)재소서양치시망막병변중대시망막신생혈관(RNV)생성적억제작용급대활성양(ROS)-핵인자κB(NF-κB)통로적영향.방법 장108지7일령C57BL/6J소서수궤평균분위3조.기중2조Smith법건립양치시망막병변모형;11일령시파리체강주사Racl-shRNA표체질립혹무의의질립,분별작위기인간예조화공백대조조.제3조우정상양배경중사양,11일령시파리체강주사Racl-shRNA표체질립작위공백간예조.15、17일령시행형광시망막포편,관찰시망막혈관발육급신생혈관정황.17일령시계수안구절편중돌파내계막적혈관내피세포핵수.17일령시진행원위잡교법화형광실시정량역전록-취합매련반응법검측소서시망막Racl화NF-κB p65아단위적mRNA함량;면역조직화학화단백질면역인기검측Racl화NF-κB p65적단백표체.결과 기인간예조시망막Racl적mRNA수평교공백대조조명현하조(t=4.500,P=0.001).여공백대조조비교,기인간예조시망막포편중무관주구、형광삼루화신생혈관총명현감경,돌파내계막적혈관내피세포핵현저감소(t=6.521,P<0.001);시망막NF-κB p65적핵역위수평명현하강(t=16.008,P<0.001),동시mRNA표체역명현하조(t=3.354,P=0.006),여Racl mRNA표체정정상관(r=0.580,P=0.012).결론 소서파리체강주사지질체포과적Racl-shRNA표체질립가유효침묵시망막Racl기인표체,조단ROSNF-κB통로이삼여억제상대결양상태하RNV생성.
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.