摘要目的 观察玻璃体腔注射慢病毒介导环腺苷酸反应成分结合蛋白1(CREB1)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 构建针对小鼠靶基因CREB1 siRNA载体,筛选并进行慢病毒包装.将140只C57BL/6J小鼠均分为正常对照组、氧诱导视网膜病变(OIR)模型组、空载体组和CREB1干扰组.正常对照组小鼠在正常空气中饲养.OIR模型组、空载体组和CREB1干扰组小鼠均于出生后7d建立OIR模型.空载体组及CREB1干扰组小鼠于出生后5d分别行玻璃体腔注射慢病毒-绿色荧光蛋白(GFP)空载体和CREB1-RNA干扰慢病毒载体1.0μl.利用突破内界膜的新生血管内皮细胞核数和荧光血管造影视网膜铺片评价视网膜新生血管情况;计算小鼠视网膜新生血管和无灌注区面积;逆转录聚合酶链反应和蛋白质免疫印迹法检测小鼠视网膜CREB1 mRNA和磷酸化CREB1 (P-REB1)蛋白表达,以及血管内皮生长因子(VEGF)-A、丝氨酸/苏氨酸蛋白激酶(Akt)、磷脂酰肌醇3激酶(PI3K)的mRNA和蛋白表达情况.结果 OIR模型组、空载体组突破内界膜的血管内皮细胞核计数较正常对照组明显增加,差异有统计学意义(P＜0.05);CREB1干扰组突破内界膜的血管内皮细胞核计数较OIR模型组、空载体组明显减少,差异有统计学意义(P＜0.05).OIR模型组、空载体组小鼠视网膜新生血管面积和无灌注区面积均较正常对照组明显增大,CREB1干扰组小鼠视网膜新生血管面积和无灌注区面积均较OIR模型组和空载体组明显缩小.4组小鼠视网膜新生血管面积和无灌注区面积比较,差异均有统计学意义(F=67.220、110.090,P＜0.05).OIR模型组、空载体组小鼠视网膜CREB1 mRNA和P-CREB蛋白表达以及VEGF-A、Akt、PI3K mRNA和蛋白表达均较正常对照组明显增加;CREB1干扰组小鼠视网膜CREB1 mRNA和P-CREB蛋白表达以及VEGF-A、Akt、PI3K mRNA和蛋白表达较OIR模型组、空载体组明显下降.4组小鼠视网膜CREB1、VEGF-A、Akt、PI3K mRNA表达比较,差异均有统计学意义(F-6.087、5.464、6.191、8.627,P＜0.05).4组小鼠视网膜P-CREB1、VEGF-A、Akt、PI3K蛋白表达比较,差异也有统计学意义(F=162.944、13.861、19.710、22.827,P＜0.05).结论 玻璃体腔注射慢病毒介导的CREB1 siRNA转染视网膜可有效抑制氧诱导小鼠视网膜新生血管的形成.

abstractsObjective To observe the inhibitory effect of lentivirus mediated small interference RNA (siRNA) targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) on retinal neovascularization (RNV) in mice.Methods CREB1 siRNA construct was created,screened and packaged to produce CREB1 RNAi-lentivirus.One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into 4 groups including normal group,oxygen induced retinopathy (OIR) group,empty vector group and CREB1 therapy group with 35 mice in each group.Mice in the normal group were kept in normal room air,while in the other three groups retinal neovascularization was induced by hypoxia on postnatal day 7 (P7).The mice in the OIR group were not treated.The mice in the vector group received intravitreal injection of lentivirus-green fluorescent protein (lenti-GFP,1 μl),and the CREB1 therapy group received CREB1 RNAi-lentivirus (1 μl) on P5.The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography.The areas of RNV and non-perfusion region were calculated.The expression of CREB1,phosphorylated-CREB1 (P-CREB1) and vascular endothelial growth factor (VEGF)-A levels,Akt and phosphoinositide 3-kinases (PI3K) in retinas were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot.Results The number of vascular cell nuclei breaking through the ILM of the OIR group and the empty vector group increased significantly compared with the normal group (P＜0.05),while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group(P＜0.05).The area of RNV and non-perfusion region of the OIR group and the empty vector group increased significantly compared with the normal group,while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group.The difference of area of RNV and non-perfusion region among 4 groups were significant (F=67.220,110.090; P＜0.05).The mRNA expression of CREB1 and protein expression of P-CREB1,the mRNA and protein expression of VEGF-A,Akt,PI3K in the retina were increased significantly in the OIR group and the empty vector group as compared with the normal group,while decreased significantly in the CREB1 therapy group as compared with the OIR group and the empty vector group.The difference of mRNA expression of CREB1,VEGF-A,Akt,PI3K in the retina among 4 groups were significant (F=6.087,5.464,6.191,8.627; P＜0.05).The protein expression of P-CREB1,VEGF-A,Akt,PI3K in the retina among 4 groups were significant (F=162.944,13.861,19.710,22.827; P＜0.05).Conclusion RNV in the mice is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1 down-regulation.