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  

Influence of prolyl hydroxylase 2 expression on endothelial barrier dysfunction induced by high glucose in humanretinal vascular endothelial cells

作　　者：梁小玲[1] 魏丽清[1] 周焕娇[1] 孙刚[1] 杨诚[1] 张熙芳[1] 杨璐[1] 林佩蓉[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广州510060

出　　处：《中华眼底病杂志》2013年第1期35-39,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：广东省科技汁划项目（20098060700106）;广州市科技计划项目（11C22060787）

摘　　要：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5mmol／L葡萄糖，甘露醇对照组细胞培养液含5mmol／L葡萄糖和25mmol／L甘露醇，高糖组细胞培养液含30mmol／L葡萄糖。培养24、48h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la（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－1a、VEGF及occludin基凶的转录水平；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-1a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-1a：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-1a、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；HIFla：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HRECsPHD2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-1a、VEGF有关。Objective To observe the influence of prolyl hydroxylase 2 （PHD2） expression on endothelial barrier dysfunction induced by high glucose in human retinal vascular endothelial ceils （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-1a （HIF-1a） 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-1a, 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-1a 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-1a：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-1a, VEGF and occludin in mannitol control group and high glucose group increased （PHD2：F=5.69, 14.27; P〈0.05. HIF-1a,：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 endothelia

关 键 词：溶胶原脯氨酸二氧酶 拮抗剂和抑制剂 血管内皮生长因子类 动物实验 

分 类 号：R77[医药卫生—眼科] R587[医药卫生—临床医学]