中华眼底病杂志

中华眼底病杂志

视网膜Müller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

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目的观察视网膜Müller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Müller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。CKO小鼠(CKO组)、NKO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(x2=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(28.31±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-1α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Müller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。

ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller 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-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring 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) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.

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,α亚基

Key words: Microglia; Mice, knockout; Vascular endothelial growth factors; Disease models, animal; Hypoxia-inducible factor 1, alpha subunit

引用本文: 金姬, 卢一, 冯佳, 任艳红, 杨莉丽, 顾伟忠. 视网膜Müller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. 中华眼底病杂志, 2017, 33(5): 508-512. doi: 10.3760/cma.j.issn.1005-1015.2017.05.016 复制

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