小干扰 RNA 靶向血管内皮生长因子基因可有效减轻小鼠模型的视网膜新生血管化。

Small interference RNA targeting vascular endothelial growth factor gene effectively attenuates retinal neovascularization in mice model.

机构信息

Tianjin Eye Hospital; Tianjin Key Lab of Ophthalmology and Visual Science; Tianjin Eye Institute; Clinical College of Ophthalmology, Tianjin Medical University, Tianjin 300020, China (Email:

出版信息

Chin Med J (Engl). 2013;126(8):1440-4.

PMID:23595374

Abstract

BACKGROUND

The mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).

METHODS

In vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified.

RESULTS

In vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals.

CONCLUSIONS

VEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

摘要

背景

视网膜新生血管的机制尚未完全阐明。许多生长因子参与了视网膜新生血管的过程，如血管内皮生长因子（VEGF）和色素上皮衍生因子（PEDF），它们分别是血管生成和抗血管生成分子的代表。氧诱导的视网膜病变（OIR）是研究视网膜新生血管的一种有用模型。本研究旨在通过调节 VEGF 与 PEDF 的比值（VEGF/PEDF），用针对 VEGF 基因的小干扰 RNA（siRNA）来抑制氧诱导的视网膜病变（OIR），从而探讨其可行性。

方法

在体外，将 EOMA 细胞与 VEGF-siRNA（psi-HI(TM)/EGFP/VEGF siRNA）和 Lipofectamine(TM) 2000 转染 24、48 和 72 小时，分别通过实时聚合酶链反应（PCR）评估 VEGF mRNA 的表达，通过 Western 印迹分析 VEGF 蛋白的水平。在体内，建立 OIR 模型小鼠，C57BL/6J 小鼠接受玻璃体腔内注射 1µl 的 psi-HI(TM)/EGFP/VEGF siRNA 和 Lipofectamine 2000 的混合物。通过 Western 印迹测量视网膜 VEGF 和 PEDF 蛋白的表达，通过荧光素血管造影观察和量化视网膜新生血管。

结果

体外 psi-HI(TM)/EGFP/VEGF siRNA 处理显著降低了 VEGF mRNA 和蛋白的表达。在体内，随着 VEGF 和 VEGF-PEDF 比值的降低，观察到干预动物的新生血管丛、无血管区、扭曲和扩张的血管明显减少。

结论

VEGF 在 OIR 中起重要作用，VEGF-siRNA 的转染可有效下调体内 VEGF 的表达，同时下调 VEGF-PEDF 比值，观察到视网膜新生血管也同时受到抑制。这些发现表明，VEGF/PEDF 可能作为视网膜新生血管治疗的潜在靶点，RNA 干扰靶向 VEGF 表达可能是一种有前途的治疗策略。