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血管内皮细胞中血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用。然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚。人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12-24h后分别用实时定量PCR和Westernblot的方法来检测VEGFmRNA的表达及ERK1/2和p38激酶的磷酸化水平。分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测。业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制。本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用。p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加。这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用。
The increased synthesis of vascular endothelial growth factor (VEGF) in vascular endothelial cells plays a very important role in promoting angiogenesis. However, the mechanism of intracellular signal transduction by hypoxia-induced VEGF secretion is not yet clear. Human umbilical vein endothelial cell line (ECV304) was cultured in hypoxia or normoxia for 12-24 h. The expression of VEGF mRNA and the phosphorylation of ERK1 / 2 and p38 kinase were detected by real-time PCR and Western blot respectively. VEGF protein secreted into the culture fluid was detected by enzyme-linked immunosorbent assay (ELISA). It has been reported that ERK inhibitor PD98059 can inhibit the hypoxia-inducible expression of VEGF gene. According to this report, we found that under hypoxia, the increase of ERK1 / 2 phosphorylation in ECV304 cells and the increase of VEGF synthesis also Can be inhibited by PD98059. The new discovery of this experiment is the role of p38 kinase activation in hypoxia-induced increase in VEGF synthesis. SB202190, an inhibitor of p38 kinase, inhibits hypoxia-induced increases in VEGF synthesis. For the first time, these data directly confirm the role of p38 kinase in hypoxia-induced increase of VEGF secretion by human endothelial cells.