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)/VEGF siRNA].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1 μl LipofectamineTM2000和空质粒psi-HITM/EGFP,3个siRNA组中分别在培养孔板内加入1μlLipofectamineTM2000+40、50、60 nmol/L质粒psi-HITM/EGFP/VEGF siRNA.分别利用实时定量PCR(real-time PCR)和Western blot法检测EOMA细胞中VEGF mRNA和蛋白的表达,以筛选最佳干扰效率的VEGF siRNA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12 d(P12)小鼠玻璃体腔内分别注射psi-HITM/EGFP和LipofectamineTM2000各0.5 μl混合物及psi-HITM/EGFP/VEGF siRNA(60 nmol/L)和LipofectamineTM 2000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-time PCR和Western blot法检测视网膜中VEGF mRNA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、60 nmol/L siRNA转染组EOMA细胞中VEGF mRNA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VEGF mRNA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＜0.001),以60 nmol/L siRNA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VEGF mRNA的相对表达量分别为1.23±0.18、4.02±0.16、3.98±0.19、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VEGF mRNA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VEGF mRNA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.
揹景 特異性地抑製視網膜中血管內皮生長因子(VEGF)的過度錶達,有望成為有效抑製新生血管的方法.RNA榦擾(RNAi)可高效、特異地抑製體內特定基因的錶達,眼跼部應用小榦擾RNA(siRNA)對身體其他組織基因的榦擾小.目的 探討在小鼠氧誘導視網膜病變(OIR)模型中VEGF的siRNA對視網膜中VEGF和新生血管的抑製作用.方法 設計併體外閤成錶達VEGFsiRNA的質粒[psi-HITM/增彊型綠色熒光蛋白(EGFP)/VEGF siRNA].小鼠血管瘤內皮(EOMA)細胞在不含抗生素的DMEM培養液中進行培養,細胞融閤達70％時分為5箇組:空白對照組培養孔培養基中無任何添加物,陰性對照組培養孔闆內加入1 μl LipofectamineTM2000和空質粒psi-HITM/EGFP,3箇siRNA組中分彆在培養孔闆內加入1μlLipofectamineTM2000+40、50、60 nmol/L質粒psi-HITM/EGFP/VEGF siRNA.分彆利用實時定量PCR(real-time PCR)和Western blot法檢測EOMA細胞中VEGF mRNA和蛋白的錶達,以篩選最佳榦擾效率的VEGF siRNA濃度.64隻齣生7d的C57BL/6J小鼠採用隨機數字錶法隨機分為正常對照組(在正常氧環境中餵養)、模型對照組(不進行玻璃體腔內註射)、空質粒組和siRNA組,其中後3箇組小鼠在氧體積分數(75±2)％的氧箱內飼養5d,然後置于正常氧環境中製備OIR動物模型.空質粒組和siRNA組于齣生後12 d(P12)小鼠玻璃體腔內分彆註射psi-HITM/EGFP和LipofectamineTM2000各0.5 μl混閤物及psi-HITM/EGFP/VEGF siRNA(60 nmol/L)和LipofectamineTM 2000各0.5μl混閤物,各組P17小鼠經高相對分子質量熒光素(FITC-dextran)灌註後製備視網膜鋪片、視網膜切片和視網膜標本,分彆觀察視網膜新生血管的變化及突破內界膜新生血管內皮細胞數,應用real-time PCR和Western blot法檢測視網膜中VEGF mRNA及其蛋白的錶達.結果 體外細胞轉染實驗錶明,空白對照組、陰性對照組及40、50、60 nmol/L siRNA轉染組EOMA細胞中VEGF mRNA和蛋白錶達的總體比較差異均有統計學意義(F=148.890,P＜0.001；F=306.960,P＜0.001),各濃度siRNA組細胞中VEGF mRNA錶達均明顯低于空白對照組,差異均有統計學意義(t＝73.950、119.890、156.480,均P＜0.001).各濃度siRNA組細胞中VEGF蛋白的錶達明顯低于空白對照組,差異均有統計學意義(t=15.452、23.344、42.119,均P＜0.001),以60 nmol/L siRNA組作用最彊.動物實驗結果錶明,與模型對照組和空質粒組比較,siRNA組小鼠視網膜無灌註區和新生血管叢明顯減少,突破內界膜血管內皮細胞的細胞覈數減少.正常對照組、模型對照組、空質粒組和siRNA組小鼠視網膜中VEGF mRNA的相對錶達量分彆為1.23±0.18、4.02±0.16、3.98±0.19、1.98±0.12,總體差異有統計學意義(F=369.780,P＜0.001),其中模型對照組和空質粒組VEGF mRNA錶達明顯高于正常對照組,差異均有統計學意義(t=37.880、37.336,P＜0.001),而siRNA組小鼠視網膜VEGF mRNA的錶達較正常對照組下降50.8％,差異有統計學意義(t=10.183,P＜0.001),siRNA組小鼠視網膜VEGF蛋白的錶達量較模型對照組下調68.0％,差異有統計學意義(t=11.473,P＜0.001),但仍高于正常對照組,差異有統計學意義(t=2.422,P＜0.001).結論 VEGFsiRNA可在體外和體內有效下調視網膜中VEGF的錶達,抑製視網膜新生血管的形成.
배경 특이성지억제시망막중혈관내피생장인자(VEGF)적과도표체,유망성위유효억제신생혈관적방법.RNA간우(RNAi)가고효、특이지억제체내특정기인적표체,안국부응용소간우RNA(siRNA)대신체기타조직기인적간우소.목적 탐토재소서양유도시망막병변(OIR)모형중VEGF적siRNA대시망막중VEGF화신생혈관적억제작용.방법 설계병체외합성표체VEGFsiRNA적질립[psi-HITM/증강형록색형광단백(EGFP)/VEGF siRNA].소서혈관류내피(EOMA)세포재불함항생소적DMEM배양액중진행배양,세포융합체70％시분위5개조:공백대조조배양공배양기중무임하첨가물,음성대조조배양공판내가입1 μl LipofectamineTM2000화공질립psi-HITM/EGFP,3개siRNA조중분별재배양공판내가입1μlLipofectamineTM2000+40、50、60 nmol/L질립psi-HITM/EGFP/VEGF siRNA.분별이용실시정량PCR(real-time PCR)화Western blot법검측EOMA세포중VEGF mRNA화단백적표체,이사선최가간우효솔적VEGF siRNA농도.64지출생7d적C57BL/6J소서채용수궤수자표법수궤분위정상대조조(재정상양배경중위양)、모형대조조(불진행파리체강내주사)、공질립조화siRNA조,기중후3개조소서재양체적분수(75±2)％적양상내사양5d,연후치우정상양배경중제비OIR동물모형.공질립조화siRNA조우출생후12 d(P12)소서파리체강내분별주사psi-HITM/EGFP화LipofectamineTM2000각0.5 μl혼합물급psi-HITM/EGFP/VEGF siRNA(60 nmol/L)화LipofectamineTM 2000각0.5μl혼합물,각조P17소서경고상대분자질량형광소(FITC-dextran)관주후제비시망막포편、시망막절편화시망막표본,분별관찰시망막신생혈관적변화급돌파내계막신생혈관내피세포수,응용real-time PCR화Western blot법검측시망막중VEGF mRNA급기단백적표체.결과 체외세포전염실험표명,공백대조조、음성대조조급40、50、60 nmol/L siRNA전염조EOMA세포중VEGF mRNA화단백표체적총체비교차이균유통계학의의(F=148.890,P＜0.001；F=306.960,P＜0.001),각농도siRNA조세포중VEGF mRNA표체균명현저우공백대조조,차이균유통계학의의(t＝73.950、119.890、156.480,균P＜0.001).각농도siRNA조세포중VEGF단백적표체명현저우공백대조조,차이균유통계학의의(t=15.452、23.344、42.119,균P＜0.001),이60 nmol/L siRNA조작용최강.동물실험결과표명,여모형대조조화공질립조비교,siRNA조소서시망막무관주구화신생혈관총명현감소,돌파내계막혈관내피세포적세포핵수감소.정상대조조、모형대조조、공질립조화siRNA조소서시망막중VEGF mRNA적상대표체량분별위1.23±0.18、4.02±0.16、3.98±0.19、1.98±0.12,총체차이유통계학의의(F=369.780,P＜0.001),기중모형대조조화공질립조VEGF mRNA표체명현고우정상대조조,차이균유통계학의의(t=37.880、37.336,P＜0.001),이siRNA조소서시망막VEGF mRNA적표체교정상대조조하강50.8％,차이유통계학의의(t=10.183,P＜0.001),siRNA조소서시망막VEGF단백적표체량교모형대조조하조68.0％,차이유통계학의의(t=11.473,P＜0.001),단잉고우정상대조조,차이유통계학의의(t=2.422,P＜0.001).결론 VEGFsiRNA가재체외화체내유효하조시망막중VEGF적표체,억제시망막신생혈관적형성.
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,1 μl LipofectamineTM 2000 + 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.
