高糖诱导人视网膜血管内皮细胞脯氨酸羟化酶2表达变化和对内皮细胞屏障功能的影响

摘要

Objective To observe the influence of prolyl hydroxylase 2 (PHD2) expression on endothelial barrier dysfunction induced by high glucose in human retinal vascular endothelial cells (HRECs).Methods The HRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose,5 mmol/L glucose +25 mmol/L mannitol,30 mmol/L glucose) as normal control group,mannitol control group and high glucose group,respectively.After the cells cultured for 24 and 48 hours,the protein levels of PHD2,hypoxia-inducible factor-1α (HIF-1α) and occludin was detected by Western blot; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by enzyme-linked immuno sorbent assay (ELISA); the transcription levels of PHD2,HIF-1α,VEGF and occludin were determined by the reverse-transcription polymerase chain reaction (RT-PCR) ; the paracellular permeability between endotheliums was detected by 7 × 104 molecular weight FITC-dextran.Results Compared with normal control group,the protein level of PHD2 in mannitol control group and high glucose group firstly decreased and then increased,the protein level of HIF-1α increased while that of occludin decreased; the secretion of VEGF increased in high glucose group but not in mannitol control group (PHD2:F=7.618,8.627; P＜0.05.HIF-1α:x2=7.692,7.652; P＜0.05.occludin:F=23.23,7.317; P＜0.05.VEGF:F=10.768,4.562; P＜0.05).Compared with normal control group,the mRNA levels of PHD2,HIF-1α,VEGF and occludin in mannitol control group and high glucose group increased (PHD2:F=5.69,14.27; P＜0.05.HIF-1α:F=6.07,10.47; P＜0.05.VEGF:F=12.31,9.14; P＜0.05.occludin:F=8.77,8.00; P＜0.05).Compared with normal control group,the paracellular permeability of mannitol control group and high glucose group increased (x2 =20.57,F=56.09; P＜0.05).Conclusions High glucose induced altered expression of PHD2 which might play an important role in endothelial barrier dysfunction.The mechanism might be associated with HIF-1α and VEGF.%目的 观察高糖诱导人视网膜血管内皮细胞(HRECs)脯氨酸羟化酶2(PHD2)的表达变化和对内皮细胞屏障功能的影响.方法 将培养至融合的HRECs分为三组,分别为正常对照组、甘露醇对照组与高糖组.正常对照组细胞培养液含5 mmol/L葡萄糖,甘露醇对照组细胞培养液含5 mmol/L葡萄糖和25 mmol/L甘露醇,高糖组细胞培养液含30 mmol/L葡萄糖.培养24、48 h后收集细胞蛋白、上清液及RNA.蛋白免疫印迹法检测细胞PHD2、缺氧诱导因子-1a(HIF-1a)及紧密连接蛋白occludin的蛋白表达水平；酶联免疫吸附试验检测细胞上清液中血管内皮生长因子(VEGF)的含量；实时荧光定量聚合酶链反应检测细胞PHD2、HIF-1α、VEGF及occludin基因的转录水平；相对分子质量7×104的异硫氰酸荧光素标记的葡聚糖检测细胞旁通透性.结果 与正常对照组相比,甘露醇对照组与高糖组HRECs中PHD2蛋白表达水平均先降低后升高,HIF-1α表达升高,occludin表达降低；高糖组细胞上清液中VEGF含量增加,差异均有统计学意义(PHD2:F=7.618、8.627,P＜0.05;HIF-1α:x2=7.692、7.652,P＜0.05；occludin:F=23.23、7.317,P＜0.05；VEGF:F=10.768、4.562,P＜0.05).与正常对照组相比,甘露醇对照组与高糖组HRECs的PHD2、HIF-1α、VEGF及occludin基因转录水平升高,差异均有统计学意义(PHD2:F=5.69、14.27,P＜0.05;HIF-1α:F=6.07、10.47,P＜0.05; VEGF:F=12.31、9.14,P＜0.05;occludin:F=8.77、8.00,P＜0.05).与正常对照组相比,甘露醇对照组与高糖组细胞旁通透性增加,差异有统计学意义(x2=20.57、F=56.09,P＜0.05).结论 高糖诱导HRECs PHD2表达发生变化.PHD2可能在内皮细胞屏障破坏中发挥一定的调控作用,其机制与HIF-lα、VEGF有关.