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目的评价脂质体介导的内皮抑素(ES)基因转移抑制缺氧诱导的小鼠视网膜新生血管的效果。探讨基因转移抑制视网膜新生血管的可行性。方法制备阳离子脂质体及PCDNA3ES复合物。选1周龄C57Bl/6N小鼠置于氧浓度为(75±2)%的氧箱中5d。回到正常环境中诱导视网膜新生血管模型。在小鼠离开氧箱的当日,向ES注射组鼠玻璃体腔注射2μl脂质体PCDNA3ES复合物;载体对照组注射等量脂质体空白载体复合物;空白对照组小鼠注射等量PBS。采用ES抗体免疫组化方法检测ES蛋白在视网膜的表达;回到正常环境中后5d,采用荧光标记的右旋糖酐血管灌注下视网膜铺片方法观察视网膜新生血管的分布;组织学切片观察比较突破视网膜内界膜的血管内皮细胞数量;透射电镜观察ES转移对视网膜超微结构的影响。结果免疫组化检查发现ES注射组小鼠玻璃体腔注射ES后24h开始有ES表达,主要位于视网膜神经纤维层细胞中,维持至少2周仍见表达;视网膜铺片观察可见空白对照组在无灌注区边缘均可见新生血管芽及荧光渗漏。ES注射组见新生血管芽明显减少;组织学检查ES注射组较其他两组突破视网膜内界膜的细胞数量减少,差异有统计学意义;ES转移后电镜下视网膜各层超微结构未见明显改变。结论采用玻璃体腔注射方法行脂质体介导的内皮抑素基因转移可以一定程度抑制缺氧诱导的小鼠视网膜新生血管生长,对视网膜无明显的毒副作用。应进一步优化转移条件以提高治疗效果。
Objective To evaluate the effect of liposome-mediated endostatin (ES) gene transfer on hypoxia-induced retinal neovascularization in mice. To explore the feasibility of gene transfer inhibiting retinal neovascularization. Methods Preparation of cationic liposomes and PCDNA3ES complex. One week old C57Bl / 6N mice were housed in oxygen chamber at (75 ± 2)% oxygen concentration for 5 days. Return to normal environment to induce retinal neovascularization model. On the day the mice left the oxygen chamber, 2 μl of lipofectamine PCDNA3ES complex was injected into the vitreous of ES injection group; the vector control group was injected with the same amount of lipofectamine; the blank control group was injected with the same amount of PBS. ES protein was detected by immunohistochemistry in the retina; 5 days after returning to normal environment, retinal neovascularization was observed by fluorescein-labeled dextran perfusion; histological sections were observed and compared to break through the retina The number of vascular endothelial cells in the membrane and the effect of ES metastasis on the ultrastructure of the retina were observed by transmission electron microscopy. Results Immunohistochemical examination showed that the expression of ES was detected in ES group at 24 h after ES injection, mainly in the retinal nerve fiber layer cells, which was maintained for at least 2 weeks. The expression of retinal detachment was observed in the blank control group Neighbors can be seen at the edge of neovascular buds and fluorescence leakage. The number of neovascular buds in ES injection group was significantly reduced. The number of cells in ES injection group was significantly lower than that in other two groups (P> 0.05). There was no significant difference in the ultrastructure of retinal layers after ES metastasis change. Conclusion Intravitreal injection of liposome-mediated endostatin gene transfer can inhibit retinal neovascularization induced by hypoxia to a certain extent, and has no obvious toxic and side effects on the retina. Should further optimize the transfer conditions to improve the treatment effect.