缺氧诱导因子1α反义寡核苷酸抑制早产鼠视网膜病血管内皮生长因子表达及新生血管形成的实验研究

Effect of-1α hypoxia inducible factor antisense oligonucleotides on the expression of vascular endothelial growth factor and retinal neovascularization in premature rat modle with retinopathy

目的:探讨缺氧诱导因子1α(Hypoxia inducible factor,HIF-1α)反义寡核苷酸(Antisense oligonucleotides,ASODN)对新生鼠仔视网膜病变(Retinopathy of prematurity,ROP)视网膜血管内皮生长因子(Vascular endothelial growth factor,VEGF)表达及新生血管形成的影响。方法:7d Wistar新生鼠仔分为两组,实验组(n=40)制作ROP动物模型,另设对照组(n=40)。两组分别从日龄12、14、16d开始眼内注射HIF-1αASODN或生理盐水(NS)。得到5组标本:分别为正常眼(A)、正常眼+ASODN(A1),ROP眼(B),ROP眼+ASODN(B1),ROP眼+NS(B2)。分别于12、14、17d视网膜铺片观察血管发育及增生情况,17d切片计数突破视网膜内界膜的血管内皮细胞核数,免疫组化法观察视网膜VEGF和HIF-1α蛋白水平的表达。结果:视网膜铺片显示B组于12d出现视网膜血管收缩变窄甚至闭塞,视网膜中央部大片无灌注区,14d视网膜新生血管开始形成,17d大量新生血管形成。B1组新生血管较B、B2组明显减少。突破视网膜内界膜的血管内皮细胞核数B2组与A,A1组差异有显著性,而B1组与B,B2组有显著性差异。免疫组化结果显示A,A1组毛细血管内皮细胞胞浆中有少许VEGF表达,无HIF-1α表达。B、B2组较A,A1组VEGF在胞浆和胞核中表达明显增加,HIF-1α在胞核中表达。B1组VEGF、HIF-1α在胞核中表达均较B,B2组明显减少。结论:吸入高氧回到常氧可使视网膜缺氧导致HIF-1α表达增加,从而上调VEGF表达促进新生血管形成。用HIF-1αASODN可通过抑制HIF-1α的表达,下调VEGF表达,抑制新生血管形成,可能成为ROP基因治疗的目的基因之一。

Objective：To investigate the effects of hypoxia inducible factor-1α（HIF-1α）antisense oligonucleotides（ASODN）on the expression of vascular endothelial growth factor（VEGF）and retinal neovascularization in premature rat with retinopathy.Methods：A total of 80 postnatal day 7 wistar rats were randomly assigned into two group.The experimental group（n=40）were established as ROP mode group.The control group（n=40）was exposed to room air in the same experimental condition.HIF-1αASODN was injected into rats＇left cavum vitreum at 12、14、16 d in these two groups.Some of the experimental group were injected with saline at 14、16 d.So there were five groups of specimens including：normal eye（A）,normal eye with HIF-1αASODN（A1）,ROP eye（B）,ROP eye with HIF-1α ASODN（B1）,ROP eye＋saline（B2）.The retinal vascular development and proliferation in five groups were evaluated by the retina stretch preparation of 15 newborn rats at 12、14、17 d respectively.Eyeball cross-section was staimed with hematoxylin and eosin（HE）.The nuclei of proliferative retinal vessels of five groups were counted.The expression of VEGF and HIF-1αin retina by immunohisto-chemical stain were observed among all groups.Results：In ROP group（B）the central vessels became tortuous and constricted and the central avascular area increased at 12 d.At 14 d,neovascularization was seen,peaking at 17 d.ROP eye with HIF-1αASODN（B1）presented the same as group A,but Neovascularization decreased obviously as compcured with group B.Retinal vessel shape of ROP eye＋saline（B2）is same as group B.VEGF was mainly expressed in capillary endothelium endochylema and nucleus of ganglionic layer of retina and inner nuclear layer.The expressions of VEGF were weaker in group A, A1 than in group B,B2,with significant differences（P〈0.05）.The expression of HIF-1αwas mainly seen in capillary endothe lium nucleus of ganglionic layer of retina and inner nuclear layer.No HIF-1αwere observed in group A and A1.The expressions of HIF-1αwere weaker in group B1 than in group B and B2,with significant difference（sP〈0.05）.Conclusion：Breathing in homo-concentration oxygen then stopping oxygen supply suddenly can increase expression of HIF-1αand VEGF in rat＇s retina.The positive correlation is presented between them.Injecting HIF-1αASODN into rat＇s left cavum vitreum can inhibit expression of HIF-1αand VEGF in retina,thereby inhibit retinal neovascularization.HIF-1αmight be one of the targets for the gene therapy for ROP.