视网膜Mnüer细胞条件性基因敲除血管内皮生长因子对氧诱导视网膜病变小鼠的影响  

The effect of conditional knocking out vascular endothelial growth factor gene on the mouse model of oxygen induced retinopathy

作　　者：金姬[1] 卢一 冯佳[1] 任艳红[1] 杨莉丽[1] 顾伟忠[1] 

机构地区：[1]浙江大学医学院附属儿童医院眼科,杭州310052 [2]上海交通大学附属第一人民医院

出　　处：《中华眼底病杂志》2017年第5期508-512,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金（81070704/H1201、81270973/H1201）

摘　　要：目的观察视网膜Mnüer细胞条件性基因敲除血管内皮生长因子（VEGF）对氧诱导视网膜病变（OIR）小鼠的影响。方法应用Cre－Loxp重组酶技术建立Mnüer细胞条件性基因敲除VEGF小鼠。Cre阳性为敲除VEGF（CKO）小鼠，Cre阴性为未敲除VEGF（NKO）小鼠。CK0小鼠（CKO组）、NK0小鼠（NKO组）各取20只建立OIR模型，观察两组小鼠在建模过程中的死亡率。小鼠17日龄时，行荧光血管造影视网膜铺片观察小鼠视网膜血管形态，计算无血管区面积占全视网膜面积的百分比；行视网膜石蜡切片苏木精伊红染色计数突破内界膜的血管内皮细胞核数；免疫荧光组织化学染色观察小鼠视网膜中缺氧诱导因子-1α（HIF-1α）的表达。结果OIR建模过程中，CKO组、NKO组小鼠总死亡率分别为65．00％、30．00％；两组小鼠总死亡率比较，差异有统计学意义（Х^2=4．912，P=0．027）。CKO组小鼠视网膜正常血管化推迟，可见大片无血管区，新生血管丛少见，整个视网膜单薄、无层次感；NKO组小鼠视网膜正常血管网状结构可见范围较大，血管密度较高，新生血管丛多见，荧光素渗漏较明显。CKO组、NKO组小鼠视网膜无血管区面积占全视网膜面积的百分比分别为（28．314-11．15）％、（16．82±7．23）％；两者比较，差异有统计学意义（t=2．734，P=0．014）。CKO组、NKO组小鼠平均每张切片突破内界膜的血管内皮细胞核数分别为（26．10±6．37）、（28．80±7．59）个；两者比较，差异有统计学意义（t-=2．437，P=-0．016）。CKO组、NKO组小鼠视网膜均可见HIF一1a呈阳性表达，主要位于神经节细胞层和光感受器细胞层；CKO组小鼠视网膜HIF-1α阳性表达强于NKO组。结论Mnüer细胞条件性基因敲除VEGF明显减弱新生小鼠在OIR环境中的生存能力，抑制部分视网膜新生血管的同时推迟了视网膜正常血管化。Objective To observe the effect of conditional knocking out （KO） vascular endothelial growth factor （VEGF） gene on the mouse model of oxygen induced retinopathy （OIR）. Methods The conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Miiller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days （P7） to P12 and in room air from P12 to P17 （each 20 mice for CKO and NKO, respectively）. The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin （HE） staining of retinal sections, and the expression of hypoxia-inducible factor-let （HIF- 1 α） was detected by immunofluorescence analysis. Results During the development of OIR, the mortality rate of CKO mice （65.00%） was higher than that of NKO mice （30.00%） with the significant difference （x2=4.912, P=0.027）. At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [（28.31 ± 11.15）%] was higher than NKO mice [（16.82±7.23）%] with the significant difference （t=2.734, P=0.014）. The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice （26.10± 6.37

关 键 词：小神经胶质细胞 血管内皮生长因子类 小鼠 基因敲除 疾病模型 动物 缺氧诱导 因子1 u亚基 

分 类 号：R774.1[医药卫生—眼科]