血管内皮细胞生长因子抑制剂对视网膜血管内皮细胞自噬的促进作用

目的　观察血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）抑制剂对小鼠视网膜血管内皮细胞（retinal vascular endothelial cells，RVECs）自噬水平的影响。方法　将RVECs随机分为五组:正常对照组、缺氧对照组、自噬抑制剂3-甲基腺嘌呤（3-methyladenine，3-MA）组、VEGF抑制剂（anti-VEGF）组和3-MA+anti-VEGF组。采用Western blot法检测RVECs中两种自噬标志蛋白微管相关蛋白1 轻链 3 （LC3）及Beclin-1的表达；计算绿色荧光蛋白（green fluorescent protein，GFP）阳性斑点细胞占比；透射电子显微镜下观察细胞内形成的自噬体的超微结构。结果　正常对照组、缺氧对照组、3-MA组、anti-VEGF及3-MA+anti-VEGF组的LC3-Ⅱ/LC3-Ⅰ分别为0.182±0.125、0.587±0.101、0.309±0.151、0.914±0.037及0.585±0.098；Beclin-1分别为0.205±0.035、0.590±0.120、0.425±0.082、0.842±0.087及0.607±0.022；GFP阳性斑点细胞比例分别为17.107%±3.521%、90.278%±2.684%、82.591%±4.490%、94.798%±1.760%及89.472%±3.764%。与正常对照组相比，缺氧对照组LC3-Ⅱ/LC3-Ⅰ和Beclin-1蛋白表达增高，GFP阳性斑点细胞的比例增加（均为P<0.05），同时自噬体增多。与缺氧对照组相比，3-MA组的LC3-Ⅱ/LC3-Ⅰ、Beclin-1蛋白表达水平及GFP阳性斑点细胞比率降低（均为P<0.05），同时自噬体减少；anti-VEGF组的LC3-Ⅱ/LC3-Ⅰ、Beclin-1蛋白表达水平及GFP阳性斑点细胞比率升高（均为P<0.05），同时自噬体增多。与anti-VEGF组相比，3-MA+anti-VEGF组的LC3-Ⅱ/LC3-Ⅰ、Beclin-1蛋白表达水平及GFP阳性斑点细胞比率降低（均为P<0.05），同时自噬体减少。结论　VEGF抑制剂对缺氧状态下视网膜血管内皮细胞的自噬水平有促进作用。

The promoting effect of vascular endothelial growth factor inhibitor on autophagy of retinal vascular endothelial cells

Objective　To explore the effects of vascular endothelial growth factor （VEGF） inhibitor on autophagy level of mouse retinal vascular endothelial cells （RVECs）.Methods　RVECs were randomly divided into five groups:the normal control group，hypoxia control group，autophagy inhibition group ［adding autophagy inhibitor 3-methyladenine （3-MA）］，anti-VEGF group （adding VEGF inhibitor） and 3-MA+anti-VEGF group.The two autophagy marker proteins microtubule associated protein 1 light chain 3 （LC3） and Beclin-1 were examined by Western blot.The ratio of green fluorescent protein-positive （GFP+） puncta in RVECs was counted.The ultrastructure of autophagosomes formed in cells was observed under transmission electron microscopy （TEM）.Results　The expression ratio of LC3-Ⅱ to LC3-Ⅰ was 0.182±0.125，0.587±0.101，0.309±0.151，0.914±0.037 and 0.585±0.098，respectively，while the expression level of Beclin-1 was 0.205±0.035，0.590±0.120，0.425±0.082，0.842±0.087 and 0.607±0.022 for the normal control group，hypoxia control group，3-MA group，anti-VEGF group and 3-MA+anti-VEGF group，respectively.GFP（+） cell rate was 17.107%±3.521%，90.278%±2.684%，82.591%±4.490%，94.798%±1.760% and 89.472%±3.764%，respectively.Compared with the normal control group，the expression of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 proteins in the hypoxia control group enhansed，the GFP+ cell rate increased （all P<0.05） and the formation of autophagosomes increased.Compared with the hypoxia control group，LC3-Ⅱ/LC3-Ⅰ，Beclin-1 and GFP+ cell rate in the 3-MA group decreased （all P<0.05），and the formation of autophagosomes decreased.Compared with the hypoxia control group，LC3-Ⅱ/LC3-Ⅰ，Beclin-1 and GFP+ cell rate in the anti-VEGF group increased （all P<0.05），and the formation of autophagosomes increased.Compared with the anti-VEGF group，LC3-Ⅱ/LC3-Ⅰ，Beclin-1 and GFP+ cell rate in the 3-MA+anti-VEGF group decreased （all P<0.05），and the formation of autophagosomes decreased.Conclusion　VEGF inhibitor has a promoting effect on the autophagy level of retinal vascular endothelial cells under hypoxia.