p42/p44 MAPK信号转导通路在缺氧诱导的人视网膜色素上皮细胞VEGF表达中的作用

Role of p42/p44 MAPK signal transduction pathway in VEGF expression under hypoxia in cultured human retinal pigment epithelial cells

目的 探讨p4 2 /p4 4丝裂原活化蛋白激酶(mitogen-activedproteinkinase ,MAPK)信号转导通路对缺氧诱导的人视网膜色素上皮(retinalpigmentepithelium ,RPE)细胞VEGF表达中的作用。方法 利用CoCl2 建立培养的人RPE细胞缺氧模型,用2 0μmol·L-1p4 2 /p4 4MAPK特异性阻断剂PD980 5 9处理RPE细胞,在缺氧条件下分别培养0min、5min、10min、30min、6 0min和12 0min ,用Westernblot方法检测磷酸化p4 2 /p4 4MAPK表达;培养人RPE细胞1h、3h、6h、12h和2 4h ,用酶联免疫吸附试验(en zymelinkedimmunosorbantassay ,ELISA)检测缺氧条件下RPE细胞上清中VEGF的含量。结果 缺氧刺激5min、10min、30min、6 0min和12 0min时,磷酸化p4 2 / p4 4MAPK水平逐渐增高;相应PD980 5 9处理组磷酸化p4 2 / p4 4MAPK水平降低,只可见磷酸化p4 2MAPK条带。在缺氧1h、3h、6h、12h和2 4h时,RPE细胞上清液中VEGF含量随时间逐渐增加(P <0 .0 1) ,PD980 5 9处理组VEGF含量较对照组显著减少(P <0 .0 1)。结论 p4 2 /p4

Objective To investigate the role of p42/p44 MAPK signal transduction pathway in VEGF expression under hypoxia in cultured human retinal pigment epithelial (hRPE) cells.Methods Cobalt Chloride was used to establish a model of hypoxia and hRPE cells were treated with the specific inhibitor of p42/p44 MAPK,PD98059 at the concentration of 20 μmol·L^(-1).The hRPE cells were cultured under hypoxia for 0 minute、5 minutes、10 minutes、30 minutes、60 minutes and 120 minutes.The expression of phospho-p42/p44 MAPK was determined by Western blot analysis.The amount of VEGF in the hRPE-conditioned supernatant was measured using enzyme linked immunosorbant assay at 1 hour、3 hours、6 hours、12 hours and 24 hours,respectively.Results Under hypoxia,the level of phospho-p42/p44 MAPK of hRPE was time-dependently increased.The level of phospho-p42/p44 MAPK of hRPE in the group treated with PD98059 was decreased and only the bands of phospho-p42 MAPK were visible.The amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner(P<0.01),and it was down-regulated by PD98059 under hypoxia(P<0.01).Conclusion p42/p44 MAPK signal transduction pathway could play a role in the VEGF expression induced by hypoxia in hRPE cells.