中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
9期
813-818
,共6页
王毅%巫娟%李罗翔%李娟%曾庆华%刘小虎
王毅%巫娟%李囉翔%李娟%曾慶華%劉小虎
왕의%무연%리라상%리연%증경화%류소호
血脂异常%ApoE基因缺失小鼠%视网膜/外丛状层,视网膜色素上皮%视网膜血管瘤增生%年龄相关性黄斑变性
血脂異常%ApoE基因缺失小鼠%視網膜/外叢狀層,視網膜色素上皮%視網膜血管瘤增生%年齡相關性黃斑變性
혈지이상%ApoE기인결실소서%시망막/외총상층,시망막색소상피%시망막혈관류증생%년령상관성황반변성
Dyslipidemia%Apolipoprotein E-deficient mouse%Retina/outer plexiform layer,retinal pigment epithelium%Retinal angiomatous proliferation%Age-related macular degeneration
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ApoE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Western blot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(514.06±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(21 048±1849)μm2,明显大于C57B L/6普通饮食组的(17 116±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(17 854±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(12 193±3806) μm2和(11 969±3616) tm2,明显大于C57BL/6普通饮食组的(5387±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VEGF蛋白的相对表达量(VEGF/β-actin)分别为1.51±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.
揹景 視網膜血管瘤增生(RAP)屬于新生血管性年齡相關性黃斑變性(AMD)的一種亞型,國內報道較少.目的 探討血脂異常的ApoE基因缺失(ApoE-/-)小鼠RAP情況及其機製.方法 採用隨機數字錶法將2月齡以C57BL/6為揹景的ApoE-/-小鼠24隻分為ApoE-/-高脂模型組和ApoE-/-普通飲食組,每組12隻；另用2月齡C57BL/6小鼠12隻普通飲食餵養作為對照(C57BL/6普通飲食組).ApoE-/-高脂模型組小鼠用高脂飼料餵養,普通飲食組小鼠均用普通飼料餵養.3箇組小鼠餵養4箇月後(6月齡)以過量痳醉法處死,摘取左眼毬製備視網膜組織切片,採用免疫組織化學染色法觀察併檢測3箇組小鼠視網膜色素上皮(RPE)細胞中血管內皮生長因子(VEGF)、視網膜外叢狀層(OPL)中VEGF受體-2(VEGFR-2)含量、OPL微血管密度(MVD)及微血管麵積(MVA),用小鼠右眼視網膜組織行Western blot檢測,定量3箇組小鼠視網膜中VEGF蛋白的錶達.結果 ApoE-/-高脂模型組和ApoE-/-普通飲食組視網膜OPL中MVD分彆為(20.67±3.20)箇/視野和(19.50±1.87)箇/視野,MVA分彆為(626.49±120.99) μm2和(514.06±88.83) μm2,均比C57BL/6普通飲食組的(12.50±1.87)箇/視野及(336.52±84.96) μm2明顯增高,差異均有統計學意義(P＜0.01)；ApoE-/-高脂模型組RPE細胞中VEGF麵積為(21 048±1849)μm2,明顯大于C57B L/6普通飲食組的(17 116±2023) μm2,差異有統計學意義(P＜0.01),而ApoE-/-普通飲食組RPE細胞中VEGF麵積為(17 854±2967) μm2,與C57BL/6普通飲食組比較差異無統計學意義(P＞0.05).ApoE-/-高脂模型組和ApoE-/-普通飲食組小鼠視網膜OPL內VEGFR-2麵積分彆為(12 193±3806) μm2和(11 969±3616) tm2,明顯大于C57BL/6普通飲食組的(5387±2225) μm2,差異均有統計學意義(P＜0.01);ApoE-/-高脂模型組和ApoE-/-普通飲食組小鼠視網膜中VEGF蛋白的相對錶達量(VEGF/β-actin)分彆為1.51±0.32和1.17±0.39,明顯高于C57BL/6普通飲食組的0.28±0.14,差異均有統計學意義(P＜0.01).結論 ApoE-/-小鼠視網膜中的VEGF含量及OPL中的VEGFR-2含量均增高,其視網膜OPL的MVD增加、MVA增大,與RAP髮病有關.
배경 시망막혈관류증생(RAP)속우신생혈관성년령상관성황반변성(AMD)적일충아형,국내보도교소.목적 탐토혈지이상적ApoE기인결실(ApoE-/-)소서RAP정황급기궤제.방법 채용수궤수자표법장2월령이C57BL/6위배경적ApoE-/-소서24지분위ApoE-/-고지모형조화ApoE-/-보통음식조,매조12지；령용2월령C57BL/6소서12지보통음식위양작위대조(C57BL/6보통음식조).ApoE-/-고지모형조소서용고지사료위양,보통음식조소서균용보통사료위양.3개조소서위양4개월후(6월령)이과량마취법처사,적취좌안구제비시망막조직절편,채용면역조직화학염색법관찰병검측3개조소서시망막색소상피(RPE)세포중혈관내피생장인자(VEGF)、시망막외총상층(OPL)중VEGF수체-2(VEGFR-2)함량、OPL미혈관밀도(MVD)급미혈관면적(MVA),용소서우안시망막조직행Western blot검측,정량3개조소서시망막중VEGF단백적표체.결과 ApoE-/-고지모형조화ApoE-/-보통음식조시망막OPL중MVD분별위(20.67±3.20)개/시야화(19.50±1.87)개/시야,MVA분별위(626.49±120.99) μm2화(514.06±88.83) μm2,균비C57BL/6보통음식조적(12.50±1.87)개/시야급(336.52±84.96) μm2명현증고,차이균유통계학의의(P＜0.01)；ApoE-/-고지모형조RPE세포중VEGF면적위(21 048±1849)μm2,명현대우C57B L/6보통음식조적(17 116±2023) μm2,차이유통계학의의(P＜0.01),이ApoE-/-보통음식조RPE세포중VEGF면적위(17 854±2967) μm2,여C57BL/6보통음식조비교차이무통계학의의(P＞0.05).ApoE-/-고지모형조화ApoE-/-보통음식조소서시망막OPL내VEGFR-2면적분별위(12 193±3806) μm2화(11 969±3616) tm2,명현대우C57BL/6보통음식조적(5387±2225) μm2,차이균유통계학의의(P＜0.01);ApoE-/-고지모형조화ApoE-/-보통음식조소서시망막중VEGF단백적상대표체량(VEGF/β-actin)분별위1.51±0.32화1.17±0.39,명현고우C57BL/6보통음식조적0.28±0.14,차이균유통계학의의(P＜0.01).결론 ApoE-/-소서시망막중적VEGF함량급OPL중적VEGFR-2함량균증고,기시망막OPL적MVD증가、MVA증대,여RAP발병유관.
Background Retinal angiomatous proliferation (RAP) is a subtype of neovascular age-related macular 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) μm2 and (514.06±88.83) μm2 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 cells 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)xm2 compared with C57BL/6 mice of the normal diet group (5387±2225)μm2(all at P＜0.01).The relative expression values (VEGF/β-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＜0.01).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.
