摘要
目的研究可溶性fms样酪氨酸激酶-1(sFlt-1)对小鼠视网膜新生血管的抑制作用。方法将54只C57/6J小鼠随机分为3组,每组18只。将其中2组小鼠于生后第7天开始置于体积分数75%氧环境中,5d后返回正常空气环境中以建立氧诱导的小鼠视网膜新生血管模型,于17d时玻璃体腔内分别注射1μLlenti-GFP和携带sFlt-1基因片段的重组慢病毒,设为模型对照组和lenti.sFlt-1组,18只正常空气环境中生长的小鼠玻璃体腔内注射1μL磷酸盐缓冲液,设为正常对照组。通过小鼠眼球连续切片苏木精-伊红染色和心脏荧光素灌注视网膜铺片法观察小鼠视网膜新生血管的变化情况,采用免疫组织化学染色法检测视网膜血管内皮生长因子(VEGF)和VEGF受体-2(KDR/Flk-1)的表达变化。结果经RT-PCR扩增的靶基因片段序列与GenBank中的标准序列相吻合。感染后的人RPE细胞能够表达sFlt-1蛋白。正常对照组小鼠视网膜内界膜平整,模型对照组小鼠突破内界膜血管内皮细胞核的数目为(47.26±6.76),而lenti.sFlt-1组为(5.21±1.93)个,差异有统计学意义(P<0.05)。视网膜铺片可见新生血管荧光素渗漏表现。慢病毒携带的sFlt-1基因片段转移至模型小鼠视网膜后,发现突入玻璃体腔的血管内皮细胞核数较模型对照组显著减少,并且视网膜毛细血管扩张、微血管瘤样改变、新生血管等荧光素渗漏表现亦明显减少;同时lenti.sFlt-1组VEGF的表达与模型对照组相比在视网膜神经节细胞层和内核层仍呈现强阳性染色,无明显变化,而KDR/Flk-1的阳性染色显著减少。结论慢病毒介导sFlt-1基因转移能显著抑制小鼠视网膜新生血管的发生。
Background Soluble fms-like tyrosine kinase-1（sFlt-1）,a soluble secreted variant of the vascular endothelial growth factor（VEGF）receptor-1 that possesses only its extracellular domain,is a specific endogenous inhibitor of VEGF.Research showed that sFlt-1 is involved in retinal vasculogenesis.ObjectivePurpose of this study was to determine the effect of sFlt-1 gene on retinal neovascularization in vivo.MethodsLentiviral expression plasmid carrying the sFlt-1 gene fragment was constructed by RT-PCR and co-transfected HEK293 T cells by calcium phosphate-DNA coprecipitation method to obtain the recombinant lenti sFlt-1.Retinal neovascularization models were induced by exposing 36 C57BL/6J mice aged postnatal day 7（P7）to the hyperoxia（75%O2）environment for 5 days and then returning to normoxia（21%O2）.Other 18 normal matched mice were raised in normoxia environment.1μL lenti-GFP or lenti.sFlt-1（with the virus titer 5×108 TU/mL）was intravitreously injected in P12-day mice in lenti-GFP group or lenti.sFlt-1 group（18 mice for each group）respectively,and the same volume of PBS was used at the same way in normal control group.The animals were sacrificed in P17 and hematoxylin-eosin staining was used to calculate the number of vascular endothelial cells nuclei breaking retinal inner limiting membrane.Retinal neovascular formation was observed in P17 mice after infusion of left ventricle of FITC-dextran under the fluorescence microscope.The expression of VEGF and KDR/Flk-1 in retinal tissue was detected by immunochemistry.The use of experimental animals followed the Standard of Association for Research in Vision and Ophthalmology.ResultsThe sequence of target gene fragment amplified by RT-PCR was consistent with standardized one in GenBank.sFlt-1 protein was expressed in human RPE cells after infected.The numbers of vascular endothelial cells nuclei breaking retinal inner limiting membrane were（47.26±6.76）in model group and（5.21±1.93）in lentivirus sFlt-1 group,showing a significant difference between them（P0.05）.Many new blood vessels and leakage of fluorescine were seen in model mice,however,the new vessels were decreased in lentivirus sFlt-1 group under the fluorescence microscope.The expressions of VEGF and KDR/Flk-1 was obviously upregulated in model group compared with normal control group,but those in lenti.sFlt-1 group showed a low expression in comparison with normal control group.ConclusionLentivirus-mediated sFlt-1 gene inhibits retinal neovascularization in anoxia mouse.
出处
《眼科研究》
CSCD
北大核心
2010年第11期1029-1034,共6页
Chinese Ophthalmic Research
基金
上海市登山计划资助项目(064119543)
