血脂异常ApoE基因缺失小鼠视网膜血管瘤增生及其机制研究

A study on retinal angiomatous proliferation and its mechanism in apolipoprotein E-deficient mouse with dyslipidemia

背景视网膜血管瘤增生（RAP）属于新生血管性年龄相关性黄斑变性（AMD）的一种亚型，国内报道较少。目的探讨血脂异常的却OE基因缺失（ApoE-／-）小鼠RAP情况及其机制。方法采用随机数字表法将2月龄以C57BL／6为背景的ApoE-／-小鼠24只分为ApoE-／-高脂模型组和ApoE-／-普通饮食组，每组12只；另用2月龄C57BL／6小鼠12只普通饮食喂养作为对照（C57BL／6普通饮食组）。ApoE-／-高脂模型组小鼠用高脂饲料喂养，普通饮食组小鼠均用普通饲料喂养。3个组小鼠喂养4个月后（6月龄）以过量麻醉法处死，摘取左眼球制备视网膜组织切片，采用免疫组织化学染色法观察并检测3个组小鼠视网膜色素上皮（RPE）细胞中血管内皮生长因子（VEGF）、视网膜外丛状层（OPL）中VEGF受体-2（VEGFR-2）含量、OPL微血管密度（MVD）及微血管面积（MVA），用小鼠右眼视网膜组织行Westernblot检测，定量3个组小鼠视网膜中VEGF蛋白的表达。结果ApoE-／-高脂模型组和ApoE-／-普通饮食组视网膜OPL中MVD分别为（20．67±3．20）个／视野和（19．50±1．87）个／视野，MVA分别为（626．49±120．99）μm2和（514．064-88．83）μm2，均比C57BL／6普通饮食组的（12．50±1．87）个／视野及（336．52±84．96）μm2明显增高，差异均有统计学意义（P〈0．01）；ApoE-／-高脂模型组RPE细胞中VEGF面积为（21048±1849）μm2，明显大于C57BL／6普通饮食组的（171164-2023）μm2，差异有统计学意义（P〈0．01），而ApoE-／-普通饮食组RPE细胞中VEGF面积为（178544-2967）μm2，与C57BL／6普通饮食组比较差异无统计学意义（P〉0．05）。ApoE-／-高脂模型组和ApoE-／-普通饮食组小鼠视网膜OPL内VEGFR-2面积分别为（12193±3806）μm2和（11969±3616）μm2，明显大于C57BL／6普通饮食组的（53874-2225）μm2，差异均有统计学意义（P〈0．01）；ApoE-／-高脂模型组和ApoE-／-普通饮食组小鼠视网膜中VECF蛋白的相对表达量（VEGF／β-aetin）分别为1．514-0．32和1．17±0．39，明显高于C57BL／6普通饮食组的0．28±0．14，差异均有统计学意义（P〈0．01）。结论ApoE-／-小鼠视网膜中的VEGF含量及OPL中的VEGFR-2含量均增高，其视网膜OPL的MVD增加、MVA增大，与RAP发病有关。

Background Retinal angiomatous proliferation （RAP） is a subtype of neovascular age-related maeular degeneration （AMD）. There are currently very few studies on RAP. Objective This study was to explore the pathogenic mechanism of RAP in apolipoprotein E-deficient （ApoE-/-） mice with dyslipidemia. Methods Twenty-four 2-month-old SPF ApoE-/- mice were randomly divided into the high fat diet group and the normal diet group, and twelve 2-month-old C57BL/6 mice received the normal diet as controls. A diet with a higher content of fat was given for 4 consecutive months in the high fat diet group,and normal diet was given in the same way in the mice of the normal diet group. The mice were sacrificed at 6 months of age. The expression of vascular endothelial growth factor （VEGF） in the retinal pigment epithelial （RPE） cells, the expression of vascular endothelial growth factor receptor-2 （VEGFR-2） in the outer plexiform layer （OPL） , microvascular density （MVD） and microvascular area （MVA） in the OPL were examined by immunohistochemistry and semi-quantitatively by histopathology with the Mias 2000 Imaging Analyzer System. The expression of VEGF protein in the retina was examined by Western blot. Results The MVD in the retinal OPL were （20. 67±3.20） and （19.50-± 1.87） , respectively, in the ApoE-/- mice of the high fat diet group and the normal diet group,which were significantly higher than that （ 12.50-± 1.87） of the C57BL/6 normal diet group （all at P〈0.01 ）. MVA in the retinal OPL were （626.49 ± 120.99 ） ~m2 and （514.06~ 88.83） i.zmz in the ApoE-/- mice of the high fat diet group and the normal diet group, respectively, showing a significant increase in comparison with the （336.52±84. 96） μm2 of the C57BL/6 normal diet group （ P〈0. 01 ）. The staining area of VEGF in RPE ceils was （21 048± 1849） μm2 in the ApoE-/- mice of the high fat diet group, showing a significant increase in comparison with the （17 116±2023 ）μm2 of the C57BL/6 normal diet group. However,no significant difference was found in the staining area of VEGF between the ApoE-/- mice with normal diet group and the C57BL/6 normal diet group （ [ 17 854＋2967 ]μm2 vs. 17 116±2023 ] μm2） （P〉0. 05 ）. Significant elevation was also seen in the staining area of VEGFR-2 in the retinal OPL of the ApoE-/- mice of the high fat diet group （ 12 193＋3806）μm2 and the ApoE-/- mice of the normal diet group （ 11 969±3616）μm2 eompared with C57BL/6 mice of the normal diet group （5387±2225）μm2（ all at P〈O. 01 ）. The relative expression values （VEGF/13-actin） of VEGF in the retinas were （ 1.51 ±0. 32） and （ 1.17 ±0.39） in the ApoE-/- mice of the high fat diet group and the normal diet group,respectively,showing a significant increase in comparison with （0.28±0. 14） of the C57BL/6 normal diet group （P〈0.0l）. Conclusions The expression of VEGF and VEGFR-2 in the retinas increases in the ApoE-/ - mouse,which leads to the enlargement of MVD and MVA in the retinal OPL and subsequent RAP occurrence.