血管内皮生长因子小干扰RNA对氧诱导视网膜病变中视网膜新生血管的抑制作用

Inhibitory effects of small interference RNA targeting vascular endothelial growth factor on oxygen-induced retinal neovascularization

背景特异性地抑制视网膜中血管内皮生长因子（VEGF）的过度表达，有望成为有效抑制新生血管的方法。RNA干扰（RNAi）可高效、特异地抑制体内特定基因的表达，眼局部应用小干扰RNA（siRNA）对身体其他组织基因的干扰小。目的探讨在小鼠氧诱导视网膜病变（OIR）模型中VEGF的siRNA对视网膜中VEGF和新生血管的抑制作用。方法设计并体外合成表达VEGFsiRNA的质粒[psi—HITM／增强型绿色荧光蛋白（EGFP）／VEGFsiRNA]。小鼠血管瘤内皮（EOMA）细胞在不含抗生素的DMEM培养液中进行培养，细胞融合达70％时分为5个组：空白对照组培养孔培养基中无任何添加物，阴性对照组培养孔板内加入1μlLipofectamine TM2000和空质粒psi—HITM／EGFP，3个siRNA组中分别在培养孔板内加入1μl Lipofeetamine TM 2000＋40、50、60nmol／L质粒psi—HJTM／EGFP／VEGFsiRNA。分别利用实时定量PCR（real—timePCR）和Westernblot法检测EOMA细胞中VEGFmRNA和蛋白的表达，以筛选最佳干扰效率的VEGFsiRNA浓度。64只出生7d的C57BL／6J小鼠采用随机数字表法随机分为正常对照组（在正常氧环境中喂养）、模型对照组（不进行玻璃体腔内注射）、空质粒组和siRNA组，其中后3个组小鼠在氧体积分数（75±2）％的氧箱内饲养5d，然后置于正常氧环境中制备OIR动物模型。空质粒组和siRNA组于出生后12d（P12）小鼠玻璃体腔内分别注射psi—HI。”／EGFP和Lipofectamine…2000各0．5斗l混合物及psi—HITM／EGFP／VEGFsiRNA（60nmol／L）和LipofectamineTM2000各0．5μl混合物，各组P17小鼠经高相对分子质量荧光素（FITC—dextran）灌注后制备视网膜铺片、视网膜切片和视网膜标本，分别观察视网膜新生血管的变化及突破内界膜新生血管内皮细胞数，应用real—timePCR和Westernblot法检测视网膜中VEGFmRNA及其蛋白的表达。结果体外细胞转染实验表明，空白对照组、阴性对照组及40、50、60nmol／LsiRNA转染组EOMA细胞中VEGFmRNA和蛋白表达的总体比较差异均有统计学意义（F=148．890，P〈0．001；F=306．960，P〈0．001），各浓度siRNA组细胞中VEGFmRNA表达均明显低于空白对照组，差异均有统计学意义（t=73．950、119．890、156．480，均P〈0．001）。各浓度siRNA组细胞中VEGF蛋白的表达明显低于空白对照组，差异均有统计学意义（t=15．452、23．344、42．119，均P〈O．001），以60nmol／LsiRNA组作用最强。动物实验结果表明，与模型对照组和空质粒组比较，siRNA组小鼠视网膜无灌注区和新生血管丛明显减少，突破内界膜血管内皮细胞的细胞核数减少。正常对照组、模型对照组、空质粒组和siRNA组小鼠视网膜中VEGFmRNA的相对表达量分别为1．23-＋0．18、4．02±0．16、3．98±O．19、1．98±0．12，总体差异有统计学意义（F=369．780，P〈0．001），其中模型对照组和空质粒组VEGFmRNA表达明显高于正常对照组，差异均有统计学意义（t=37．880、37．336，P〈0．001），而siRNA组小鼠视网膜VEGFmRNA的表达较正常对照组下降50．8％，差异有统计学意义（t=10．183，P〈0．001），siRNA组小鼠视网膜VEGF蛋白的表达量较模型对照组下调68．0％，差异有统计学意义（t=11．473，P〈0．001），但仍高于正常对照组，差异有统计学意义（t=2．422，P〈0．001）。结论VEGFsiRNA可在体外和体内有效下调视网膜中VEGF的表达，抑制视网膜新生血管的形成。

Background Arresting the overexpression of vascular endothelial growth factor （VEGF） will be a new approach to the inhibition of neovascularization. RNA interference （RNAi） can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression. Objective This study was to investigate the effect of small interference RNA （siRNA） targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy （OIR） model. Methods psi-HITM/enhanced green fluorescent protein （EGFP）/VEGF siRNA was designed and prepared in vitro. Mouse endothelioma （EOMA） were cultured in DMEM without antibiotic and divided into 5 groups. The cells were incubated in DMEM only in the blank control group ;while 1 μl of LipofectamineTM 2000 ± psi-HITM/EGFP, 1μ1 LipofectamineTM2000 ± 40,50 or 60 nmol/L of psi-HITM/ EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups, respectively. The expression of VEGF mRNA and protein was detected by real time PCR （RT-PCR） and Western blot. The optimal effective concentration of VEGF siRNA was assessed. OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of （75±2） % for 5 days and then to normal air. The mice were randomized into the model group, null vector group and VEGF siRNA group. 1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA （60 nmol/L） and LipofectamineTM 2000 was intravitreally injected, respectively,in the null vector group and VEGF siRNA group. The normal mice were used as the normal control group. Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot, respectively, and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization, and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane （ ILM）. Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group, negative control group and 40,50,60 nmol/L VEGF siRNA groups （ F = 148.890,P 〈 0.001; F = 306.960, P〈0.001）, and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group （t=73. 950,119. 890,156. 480 ,all at P〈 0. 001 ）. Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group （t= 15. 452,23. 344,42. 119 ,all at P〈0. 001 ）. The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L. In vivo study determined that compared to the model group and null vector group, the non- perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group. The relative expression level of VEGF mRNA in the retinas was 1.23±0. 18,4. 02±0. 16,3.98±0. 19 and 1.98±0. 12 in the normal control group,model group, null vector group and VEGF siRNA group, respectively, with a significant difference among them （ F = 369. 780, P〈0. 001 ） , and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group （t= 37. 880,37. 336, both P〈0. 001 ） , and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8% （t= 10. 183 ,P〈0. 001 ）. The difference in the expression levels of VEGF protein also was assayed among the various groups （ F = 408. 980, P〈0. 001 ） , and VEGF level in the retina was lowered by 68.0% in the VEGF siRNA group compared to the model group （t= 11. 473,P〈0. 001 ）. However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group （t = 2. 422, P〈 0. 001 ）. Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.