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目的:研究血管内皮细胞生长因子(VEGF)对肿瘤坏死因子(TNF-α)和过氧化氢(H_2O_2)诱导生主动脉内皮细胞(BAEC)凋亡的影响及信号机制.方法:BAEC培养并传代于DMEM.经TNF-α或H~2O_2处理24h后,Hoechst 33258染色,荧光显微镜观察形态学变化及凋亡细胞计数.MTT法测定细胞活性,琼脂糖凝胶电泳分析DNA降解,Western blot法检测磷酸化p~(38)和p~(42)/p~(44) CCDPK表达.结果:TNF-α5000kU/L和H_2O_2 300μmol/L均可诱导BAEC产生DNA断片.VEGF 100 μg/L显著增强TNF-α和H_2O_2诱导的磷酸化p~(42)/p~(44) CCDPK表达,而明显抑制磷酸化 p~(38) CCDPK的活化.对二者所致 BAEC凋亡起明显的抑制作用.P~(42)/p~(44) CCDPK抑制剂U0126可取消 VEGF引起的磷酸化p~(42)/p~(44) CCDPK表达上调和其抗凋亡作用.结论:VEGF通过其共调节作用激活p~(42)/p~(44) CCDPK,抑制p~(38) CCDPK信号途径而对TNF-α和H_2O_2所致凋亡产生的抑制效应,是内皮细胞存活的重要机制.
AIM: To investigate the effect of vascular endothelial growth factor (VEGF) on the apoptosis of tumor cells induced by tumor necrosis factor (TNF-α) and hydrogen peroxide (H_2O_2) and the signal transduction mechanism.Methods: The cells were stained with Hoechst 33258 for 24 h after treatment with TNF-α or H 2 O 2, and morphological changes and apoptotic cell counts were observed by fluorescence microscopy. The cell viability was measured by MTT method. DNA degradation was analyzed by agarose gel electrophoresis and Western blot Phosphorylation of p38 and p42 / p44 were detected by Western blotting.Results: DNA fragments were induced by BAF at a concentration of 5000 kU / L and 300 μmol / L H 2 O 2, respectively.VEGF 100 μg / L significantly increased TNF α and H_2O_2 -induced phosphorylation of p42 / p44 CCDPK, and inhibited the phosphorylation of p38 CCDPK obviously, which could significantly inhibit the apoptosis of BAEC induced by both. P (42) / p ~ (44) CCDPK inhibitor U0126 could up-regulate the phosphorylation of p42 (42) / p44 CCDPK induced by VEGF and its anti-apoptosis effect.Conclusions: It is an important mechanism of endothelial cell survival that the activation of p ~ (42) / p ~ (44) CCDPK and the inhibition of p ~ (38) CCDPK signaling pathway on TNF-α and H_2O_2-induced apoptosis.