摘要
目的 通过RNA干扰技术抑制小鼠视网膜新生血管形成过程中Twist基因的表达,观察视网膜新生血管内皮细胞的迁移变化和细胞转型规律,寻找抑制视网膜新生血管发生的新靶点.方法 实验研究.取健康C57BL/6J新生小鼠建立高氧诱导小鼠视网膜新生血管动物模型.小鼠生后第12天,分别给予Twist干扰质粒和无关对照质粒玻腔注射治疗.第17天取材,行视网膜Evans蓝灌注铺片、组织病理学检查、新生血管内皮细胞计数和免疫组织化学检测及Real-Time PCR检测.对各组视网膜新生血管内皮细胞计数和Real-Time PCR检测目标基因的表达变化,采用单因素方差分析进行统计学比较.结果 光镜下观察突破内界膜的视网膜新生血管,正常对照组、高氧诱导组、干扰质粒组和对照质粒组突破内界膜的血管内皮细胞平均每眼分别为0.34±0.11、32.73±6.38、4.56±2.02和20.17±6.49.小鼠视网膜Evans蓝灌注铺片观察和石蜡切片HE染色观察显示Twist干扰质粒组小鼠第17天较高氧诱导组视网膜血管迂曲渗漏减轻,新生血管明显减少.Twist干扰质粒组视网膜新生血管内皮细胞计数较高氧诱导组显著减少.免疫组织化学检测可见Twist干扰质粒组小鼠视网膜Twist和波形蛋白表达较高氧诱导组明显减少.使用Real-Time PCR方法检测各组小鼠第17天视网膜Twist基因和波形蛋白基因的表达,Twist干扰质粒组表达较高氧诱导组下调（F=27.214,31.211;P＜0.05）.结论 在小鼠视网膜新生血管形成过程中,细胞转型调控的Twist基因发挥重要作用,通过RNA干扰技术抑制Twist基因的表达,可阻遏细胞转型的发生,抑制视网膜新生血管的形成.
Objective The purpose of this research is to find the law of neovascular endothelial cell migration and transition through repressing the expression of Twist in mouse＇s retinal neovascularization with RNAi, and get a new target of inhibit retinal neovascularization. Methods Oxygen-induced retinopathy （OIR） was produced in new bom C57BL/6J mice by exposing postnatal day 7 （P7） pups to 75% oxygen for 5 days. P12 pups were injected 1 μl pTwist. siRNA plasmid solution or 1 μl negtive siRNA plasmid solution into vitreous cavity. Eyeballs were enucleated for Evans blue angiography, histopathologic examination,neovascular endothelial cell counting, immunohistochemistry and Real-Time PCR. Results Observed by light microscopy retinal neovascularization, the number of vascular endothelial cells per eye were 0.34±0.11,32.73±6.38, 4.56±2.02 and 20.17±6.49 in the normal control group, hyperoxia group,Twist plasmid group and the control plasmid group. Mouse retinal Evans blue perfusion and HE staining of paraffin sections showed that retinal vascular leakage, tortuous and angiogenesis significantly reduced in Twist plasmid group compared with hyperoxia group. Endothelial cell count was significantly decrease in Twist plasmid group. Both immunohistochemistry and real time PCR proved that Twist and vimentin expression in hyperoxia group were significantly higher than that of Twist plasmid group （ F=27.214,31.211 ;P〈0.05）. Conclusion As mice retinal neovasculars growth, Twist may play important roles as a cell transition regulatory factor. Repressing Twist with RNAi, we can repress cell transition and inhibit retinal neovascular.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2011年第3期217-222,共6页
Chinese Journal of Ophthalmology
基金
基金项目：国家自然科学基金（30772386）
山东省自主创新重大科技专项计划基金（2006GG1102020）
山东省自然科学基金（Y2006C94）
