改良内皮抑素基因转移对碱烧伤角膜新生血管的抑制作用

Inhibitive effects of antiangiogenesis with mutant endostatin gene transforming on corneal neovascularization induced by alkali burn

目的观察含重复RGD序列的改良内皮抑素(endostatin,ES)(poly RGD-ES)基因-转移抑制碱烧伤诱导的兔角膜新生血管(corneal neovascularization,CNV)的效果,探讨其作用机制。方法将27只(54眼)兔随机平均分为3组,采用1mol.L-1NaOH碱烧伤法诱导兔CNV形成。碱烧伤后分别在结膜下注射50g.L-1pCI-poly RGD-ES转染液、50g.L-1pCI-ES转染液和PBS缓冲液各0.2mL;每周2次,共2周。动态观察CNV的生长状况,计算CNV面积。第3天、第7天、第14天利用免疫组织化学方法观察血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,ELISA法检测房水中VEGF蛋白的表达。结果pCI-poly RGD-ES转染组CNV面积7d时达高峰,为(6.368±0.739)mm2,各时间点均明显少于其他2组,差异有显著统计学意义(P<0.001);7d时pCI-poly RGD-ES转染组房水中VEGF表达为(0.644±0.082)μg.L-1,7d后开始下降,各检测点与其他2组比较,差异有显著统计学意义(P<0.001)。结论poly RGD-ES可有效抑制碱烧伤诱导的CNV的生长,活性强于ES。

Objective To investigate the effects of subconjunctival mutant endostatin gene tranforming with RGD sequence（poly RGD-ES） on corneal neovascularization （CNV） in rabbits induced by alkali burn. Methods Twenty-seven New Zeal- and white rabbits（54 eyes） were involved,and divided equally into 3 groups at random. Both eyes per rabbit were used. Alkali burn of cornea in rabbit was induced by application of 1 mol · L^-1 NaOH. 0.2 mL 50 g · L^-1 pCI-poly RGD-ES,0.2 mL mixture of 50 g · L^-1 pCI-ES and PBS buffer solution were injected into subconjunctival tissues of rabbits immediately after alkali burn, respectively, twice a week, 2 weeks in total. The growth state of CNV was observed every day, and total CNV area was assessed. Expression of vascular endothelial growth factor（VEGF） in the cornea was detected by immunohistochemistry on the 3th day, the 7th day and the 14th day, respectively. VEGF in aqueous humor was quantified by ELISA assays. Results pCI-poly RGD-ES treatment group showed smaller area of blood vessels than the other two groups, the area of CNV reached prominent（6. 358 ± 0.739） mm2 on the 7th day, there were statistical significant （P 〈 0. 001 ）. pCI-poly RGD-ES treatment group showed significant downregulation of VEGF compared with the other two groups,VEGF level was （0.644 ＋0.082） μg · L^-1on the 7th day, there were satistical significance （P 〈 0. 001 ）. Conclusion Mutant endostatin with RGD sequence could effectively inhibit the growth of CNV induced by alkali burn. and has more activity than endostatin.