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目的:研究肿瘤坏死因子(TNF-α)诱导牛主动脉内皮细胞(BAEC)凋亡及其信号途径。方法:BAEC培养并传代于DMEM。经TNF-α处理24h后,Hoechst33258染色,荧光显微镜观察形态学变化及凋亡细胞计数。MIT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p38和p44/42CCDPK表达。结果:TNF-α诱导BAEC产生典型的凋亡细胞形态学变化(核浓染,核碎裂)和DNA断片。TNF-α(100-5000kU/L)浓度依赖性诱导BAEC凋亡,并同时刺激磷酸化p44/42和p38CCDPK的表达.p44/42CCDPK抑制剂U0126可完全阻断TNF-α诱导的p44/42CCDPK的活化,显著增强TNF-α致凋亡作用;而p38 CCDPK抑制剂SB203580可完全阻断TNF-α诱导的p38CCDPK的活化,还可增强TNF-α诱导的磷酸化p44/42CCDPK的表达,明显抑制TNF-α促凋亡作用。结论:TNF-α同时激活p38和p44/42CCDPK,这两种CCDPK信号通路在TNF-α诱导BAEC凋亡中起相反作用。
Objective: To study the apoptosis and signal pathway induced by tumor necrosis factor (TNF-α) in bovine aortic endothelial cells (BAEC). Methods: BAECs were cultured and subcultured in DMEM. After treatment with TNF-α for 24 h, Hoechst33258 staining and morphological changes were observed by fluorescence microscopy. Cell viability was determined by MIT assay. DNA degradation was analyzed by agarose gel electrophoresis. Expression of phosphorylated p38 and p44 / 42CCDPK was detected by Western blot. RESULTS: TNF-αinduced BAECs to produce typical apoptosis morphological changes (nuclear staining, nuclear fragmentation) and DNA fragments. TNF-α (100-5000kU / L) induces BAEC apoptosis in a dose-dependent manner and simultaneously stimulates the expression of phosphorylated p44 / 42 and p38CCDPK.p44 / 42CCDPK inhibitor U0126 can completely block TNF-α-induced p44 / 42CCDPK Activated, and significantly enhanced the apoptosis of TNF-α. However, p38 CCDPK inhibitor SB203580 completely blocked the activation of p38CCDPK induced by TNF-α, and enhanced the expression of phosphorylated p44 / 42CCDPK induced by TNF-α -α and promote apoptosis. CONCLUSION: TNF-α activates both p38 and p44 / 42CCDPK. These two CCDPK signaling pathways play an opposite role in the apoptosis of BAEC induced by TNF-α.