Li Zhi, He Tao, DU Ke, Xing Yi-qiao
Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Email:
Zhonghua Yan Ke Za Zhi. 2013 Dec;49(12):1111-7.
To investigate the mechanism and inhibitory effects of overexpression of 15-lipoxygenase-1 inhibiting oxygen-induced retinal neovascularization in mice.
Experimental study. Eighty-eight 7-day-old C57BL/6J mice were randomly divided into the normal control group, induced model group, gene treated group and empty vector group with 22 mice in each group. The mice with their mothers were arisen in 75% ± 2% O₂ environment for 5 days and then returned to normoxia for 5 days to establish the oxygen-induced retinopathy (OIR) model. At postnatal day 12, the gene treated group was received an intravitreous injection of Ad-15-LOX-1-EGFP at 1.0 µl, while the empty vector group was received the same volume of Ad-EGFP. At postnatal day 17, real-time PCR and Western Blot methods were used to detect the mRNA and protein expression levels of 15-LOX-1, peroxisome proliferator-activated receptor γ (PPAR-γ) , vascular endothelial growth factor-A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR-2) in the retina. The relative retinal non-perfusion and neovascularization areas were evaluated by FITC-dextran fluorescein angiography on flat-mounted retina. The number of endothelial cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. Rank sum test and one-way ANOVA were used to assess statistical significance within groups.
The 15-LOX-1 and PPAR-γ mRNA and protein expression levels were higher in gene treated group (15-LOX-1: 2.17 ± 0.25, 1.45 ± 0.10;PPAR-γ:2.12 ± 0.29, 0.85 ± 0.03) than those in induced model group (15-LOX-1:0.62 ± 0.03, 0.66 ± 0.04; PPAR-γ:0.67 ± 0.18, 0.48 ± 0.03) and empty vector group (15-LOX-1:0.51 ± 0.14,0.57 ± 0.03;PPAR-γ:1.07 ± 0.09,0.52 ± 0.02) ( t15-LOX-1 = 12.511, 13.402, both P < 0.01; tPPAR-r = 9.420, 6.813, both P < 0.01). On the contrary,VEGF-A and VEGFR-2 expression levels were lower in gene treated group (
VEGF-A: 0.87 ± 0.07, 0.34 ± 0.01; VEGFR-2:1.02 ± 0.12, 0.45 ± 0.03) than those in induced model group (
VEGF-A: 3.49 ± 0.53,0.74 ± 0.04; VEGFR-2:2.28 ± 0.44, 0.82 ± 0.01) and empty vector group (
VEGF-A: 2.30 ± 0.25,0.69 ± 0.02; VEGFR-2:1.88 ± 0.16, 0.76 ± 0.03) (tVEGF-A = 10.662, 5.843, both P < 0.01; tVEGFR-2 = 6.731, 4.763, both P < 0.01). The relative retinal non-perfusion and neovascularization areas were significantly smaller, and the number of endothelial cell nuclei breaking through the ILM was obviously lower in gene treated group(5.88 ± 1.12; 9.37 ± 1.85; 1.25 ± 0.89) than those in induced model group (21.25 ± 2.87; 24.13 ± 4.29; 60.63 ± 10.82) and empty vector group (19.50 ± 1.78; 23.13 ± 3.52; 54.63 ± 7.63) (P < 0.01; P < 0.01).
Overexpression of 15-LOX-1 inhibits ORI neovascularization in mice via up-regulation of PPAR-γ and down-regulation of VEGF-A and VEGFR-2 expression.
探讨15-脂氧合酶-1过表达抑制小鼠氧诱导性视网膜新生血管形成的机制及抑制作用。
实验研究。将88只7日龄C57BL/6J小鼠随机分为正常对照组、诱导模型组、基因治疗组和空载体组，每组22只。将携带幼崽的母鼠置于75%±2%氧气环境中5天，然后恢复常氧5天，以建立氧诱导性视网膜病变（OIR）模型。在出生后第12天，基因治疗组玻璃体腔内注射1.0 μl Ad-15-LOX-1-EGFP，空载体组注射相同体积的Ad-EGFP。在出生后第17天，采用实时PCR和蛋白质印迹法检测视网膜中15-脂氧合酶-1（15-LOX-1）、过氧化物酶体增殖物激活受体γ（PPAR-γ）、血管内皮生长因子-A（VEGF-A）和血管内皮生长因子受体2（VEGFR-2）的mRNA和蛋白表达水平。通过荧光素标记葡聚糖眼底血管造影评估平铺视网膜上相对视网膜无灌注区和新生血管形成区。在苏木精-伊红染色的视网膜切片上计数突破内界膜（ILM）的内皮细胞核数量。采用秩和检验和单因素方差分析评估组内统计学意义。
基因治疗组15-LOX-1和PPAR-γ的mRNA和蛋白表达水平（15-LOX-1：2.17±0.25，1.45±0.10；PPAR-γ：2.12±0.29，0.85±0.03）高于诱导模型组（15-LOX-1：0.62±0.03，0.66±0.04；PPAR-γ：0.67±0.18，0.48±0.03）和空载体组（15-LOX-1：0.51±0.14，0.57±0.03；PPAR-γ：1.07±0.09，0.52±0.02）（t15-LOX-1=12.511，13.402，P均<0.01；tPPAR-γ=9.420，6.813，P均<0.01）。相反，基因治疗组VEGF-A和VEGFR-2的表达水平（VEGF-A：0.87±0.07，0.34±0.01；VEGFR-2：1.02±0.12，0.45±0.03）低于诱导模型组（VEGF-A：3.49±0.53，0.74±0.04；VEGFR-2：2.28±0.44，0.82±0.01）和空载体组（VEGF-A：2.30±0.25，0.69±0.02；VEGFR-2：1.88±0.16，0.76±0.03）（tVEGF-A=10.662，5.843，P均<0.01；tVEGFR-2=6.731，4.763，P均<0.01）。基因治疗组相对视网膜无灌注区和新生血管形成区明显较小，突破ILM的内皮细胞核数量明显低于诱导模型组（21.25±2.87；24.13±4.29；60.63±10.82）和空载体组（19.50±1.78；23.13±3.52；54.63±7.63）（P<0.01；P<0.01）。
15-LOX-1过表达通过上调PPAR-γ和下调VEGF-A及VEGFR-2表达抑制小鼠氧诱导性视网膜病变新生血管形成。
