自噬抑制剂3-MA抑制高糖条件下猴脉络膜视网膜血管内皮细胞血管形成

摘要

目的：研究高浓度葡萄糖对体外培养的猴脉络膜视网膜血管内皮细胞（ RF／6A）自噬水平的影响，探讨自噬抑制剂3-MA对RF／6A细胞新生血管形成的影响。方法取体外培养的生长良好的RF／6A细胞用于实验，将细胞随机分为4组，分别是对照组、低糖组（5 mmol／L的D-葡萄糖）、高糖组（25 mmol／L的D-葡萄糖）、高糖＋3-MA组（5 mM的3-MA预处理细胞后加入含25 mmol／L的D-葡萄糖培养液培养）。24 h后通过MTT法检测细胞活力，细胞划痕法检测细胞迁移，基质胶（Matrigel）法检测血管管腔形成，观察高糖对RF／6A细胞活力、迁移、管腔形成的影响及其3-MA的作用。结果①同对照组比较，低糖组RF／6A细胞活力下降，高糖组RF／6A细胞活力进一步下降（ P ＜0．05），高糖＋3-MA组细胞活力较低糖与高糖组明显提高（ P ＜0．05）；②对照组、低糖组、高糖组、高糖＋3-MA组细胞迁移面积（像素）分别是：40250±2015，44528±2273，55793±2158，39726±1983，组间差异具有统计学意义（ P ＜0．05）；③对照组、低糖组、高糖组、高糖＋3-MA组RF／6A细胞管腔形成数分别为16．9±3．3，19．8±3．6，24．7±1．9，10．9±3．3，组间差异有统计学意义（ P ＜0．05）；与对照组比较，D-葡萄糖促进RF／6A细胞管腔形成（ P ＜0．05），而3-MA能够显著抑制RF／6A细胞管腔的形成。结论高糖能够抑制RF／6A细胞活力，促进RF／6A细胞迁移和管腔形成，通过3-MA预处理可以一定程度恢复细胞活力，显著抑制高糖诱导的细胞迁移和管腔形成。提示自噬可能参与调控高糖诱导的视网膜新生血管形成。%Objective To investigate the regulation of autophagy in RF/6A cells cultured in high glucose, and to study the effect of autophagy inhibitor 3-MA on cell viability, migration and tube formation.Methods Cultured RF/6A cells were randomly divided into four groups according to glucose concentrations in culture medium:control group ( no glu-cose) , low dose ( D-glucose 5 mmol/L) , high dose ( D-glucose 25 mmol/L) and high glucose with 3-MA ( D-glucose 25 mmol/L+3-MA 5mM) .RF/6A cells were cultured for 24 hours.Cell viability was detected by MTT assay.Migration was measured by scratch assay.Tube formation was observed by seeding RF/6A cells in matrigel.Results A values were sig-nificantly ( P <0.05) different between groups.Compared to the control group, cell viability was significantly inhibited in RF/6A cultured in low dose or high dose groups.But cell viability in high glucose with 3-MA group was elevated compared to both the low dose group and high dose group.Cell migration area ( pixels) was significantly different in four groups ( P<0.05).The numbers of tube formation were also significantly different among four groups ( P <0.05).Both low and high glucose promoted tube formation, while 3-MA inhibited the tube formation despite of high glucose.Conclusion High glucose can inhibit RF/6A cell activity, promote migration and tube formation.Presence of 3-MA significantly enhanced cell viability, inhibited cell migration and tube formation in RF/6A.Our study indicated that autophagy maybe participate in regulating the formation of high glucose induced retinal neovascularization.