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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。
Objective To investigate the effect of recombinant adeno-associated virus-mediated inhibition of vascular endothelial growth factor antisense nucleotide (rAAV aVEGF165) on the occurrence and development of corneal neovascularization (CNV) in rats. Methods The rAAV aVEGF165 and the recombinant adeno-associated virus-mediated structural gene of β-galactosidase (rAAV Lacz) were constructed by triple-plasmid co-transfection. Forty-eight Sprague-Dawley rats were randomly divided into experimental and control groups by lottery, with 24 rats in each group. Alkaline burn method was used to induce CNV formation. The experimental group was injected rAAV aVEGF165 (1010 pfu / ml) into the conjunctival sac of the experimental group and rAAV Lacz (1010 pfu / ml) into the conjunctival sac of the control group. The X-gal staining of LacZ- 1, 2, 4, 7, 14 and 30 days after alkali burn. The morphological analysis was used to evaluate the growth of corneal neovascularization. The expression of VEGF in cornea was detected by immunohistochemistry. Results The expression of Lacz gene in conjunctival tissue was 21.36% ± 1.07% 2 days after injection of rAAV Lacz conjunctiva in control group. The expression efficiency of Lacz gene in subconjunctival tissue after 30 days was 28.02% ± 1.16%. The rAAV aVEGF165 was injected into the conjunctival sac of the alkali burn group. The area of CNV in the experimental group was significantly decreased, the neovascularization density was decreased and the expression of VEGF was significantly decreased in the corneal tissue (F = 1639.22, F = 2187.16, F = 719.17, P <0.01). Conclusion rAAV aVEGF165 can significantly inhibit the formation of CNV after alkali burn, the mechanism of which is related to the decrease of VEGF expression in corneal tissue. Recombinant adeno-associated virus is an effective vector for the treatment of ocular antisense gene.