血管内皮生长因子反义核苷酸抑制大鼠角膜新生血管的实验研究

目的探讨重组腺相关病毒介导的血管内皮生长因子反义核苷酸(rAAV aVEGF165)抑制大鼠角膜新生血管(CNV)发生与发展的作用。方法三质粒共转染法构建rAAV aVEGF165和重组腺相关病毒介导的β半乳糖苷酶的结构基因(rAAV Lacz)。48只SD大鼠用抽签法随机分成实验组和对照组,每组24只,碱烧伤方法诱导CNV生成。实验组结膜囊注射rAAV aVEGF165(1010pfu/ml),对照组结膜囊注射rAAV Lacz(1010pfu/ml);5溴4氯3吲哚βD半乳糖苷(X gal)组织化学染色,检测Lacz基因在结膜囊中的表达情况;碱烧伤后1、2、4、7、14及30d,形态学分析评价角膜新生血管的生长情况,免疫组织化学检测角膜组织中VEGF的表达。结果对照组rAAV Lacz结膜囊注射2d后,检测Lacz基因在结膜组织中的表达效率为21.36%±1.07%;30d后Lacz基因在结膜下组织中的表达效率为28.02%±1.16%。碱烧伤后结膜囊注射rAAV aVEGF165,实验组CNV面积明显减少,新生血管密度降低,VEGF在角膜组织中的表达明显降低,差异有统计学意义(F=1639.22,F=2187.16,F=719.17,P<0.01)。结论rAAV aVEGF165能显著抑制碱烧伤后CNV的形成,其机制与降低角膜组织中VEGF的表达有关,重组腺相关病毒是眼反义基因治疗的有效载体。

Antisense vascular endothelial growth factor suppressed corneal neovascularization in rats

OBJECTIVE: To explore whether recombinant adeno-associated virus mediated antisense vascular endothelial growth factor (rAAV-aVEGF) gene transfer inhibits the development of corneal neovascularization (CNV) in a rat model. METHODS: rAAV-aVEGF(165) and recombinant adeno-associated virus mediated Lacz (rAAV-Lacz) were constructed by three-plasmid cotransfection methods. Forty-eight Sprague-Dawley (SD) rats were divided into two groups randomly. CNV were induced in vivo by alkaline cauterization of the central cornea. Twenty-four rats were injected with rAAV-aVEGF(165) (10(10) pfu/ml) into conjunctiva; another rats were injected with rAAV-Lacz (10(10) pfu/ml) into conjunctiva as controls. The Lacz gene expression was evaluated by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) immunohistochemical staining. 1 to 30 days post-cautery, CNV was evaluated by morphometric analysis, and expression of VEGF was evaluated by immunohistochemistry. RESULTS: Two days later, 21.36% +/- 1.07% Lacz gene expression was detected in conjunctival sac in control group. 30 d later, 28.02% +/- 1.16% Lacz gene remained expression. Morphometric analysis and immunohistochemistry demonstrated rAAV-mediated antisense VEGF(165) significantly inhibited CNV. The VEGF(165) protein was decreased post-cautery compared to control group injected with. rAAV-Lacz (F = 1639.22, F = 2187.16, F = 719.17, P < 0.01). CONCLUSIONS: This study suggests that rAAV-aVEGF(165) can sufficiently inhibit the cautery-induced CNV, and the effect is associated with the inhibition of VEGF production.