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目的:研究雌二醇(17β-estradiol,E_2)对肿瘤坏死因子α(TNF-α)诱导牛主动脉内皮细胞(BAEC)凋亡的影响及机制.方法:BAEC培养并传代于DMEM.BAEC经Hoechst 33258染色后用荧光显微镜观察形态学变化及计数凋亡细胞;用MTT法测定细胞活性;用琼脂糖凝胶电泳分析DNA降解;用Westernblot法检测磷酸化p~(38)和p~(42) CCDPK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.E_2(0.1pmol/L-100 nmol/L)能增强TNF-α诱导的磷酸化p~(44)/42表达,同时抑制p~(38) CCDPK的活化而浓度依赖性阻止TNF-α诱导的BAEC凋亡;E_2 1nmol/L明显减弱TNF-α所致DNA断裂;p~(44)/42 CCDPK抑制剂U0126可拮抗E_2的作用.结论:雌激素通过激活p~(44)/42CCDPK,同时抑制p~(38) CCDPK而产生的抗凋亡效应,是其使内皮细胞存活的重要机制.
AIM: To investigate the effect and mechanism of 17β-estradiol (E_2) on the apoptosis of bovine aortic endothelial cells (BAEC) induced by tumor necrosis factor α (TNF-α) .Methods: BAEC was cultured and passaged in DMEM.BAEC Hoechst 33258 staining was observed by fluorescence microscopy morphological changes and count apoptotic cells; MTT assay cell viability; DNA degradation was analyzed by agarose gel electrophoresis; Western blot was used to detect phosphorylation of p ~ (38) and p ~ (42) ) CCDPK expression.Results: TNF-αinduced BAECs to produce typical apoptosis morphological changes (nuclear staining, nuclear fragmentation) and DNA fragments.E2 (0.1 pmol / L-100 nmol / L) Induced the phosphorylation of phosphorylation of p44 and expression of p38 and inhibited the activation of p38 CCDPK in a concentration-dependent manner, which prevented the apoptosis of BAEC induced by TNF-α. E_2 1 nmol / L significantly attenuated the DNA fragmentation induced by TNF-α ; p ~ (44) / 42 CCDPK inhibitor U0126 can antagonize the effect of E_2.Conclusion: The anti-apoptotic effect of estrogen by p ~ (44) / 42CCDPK and p38 CCDPK An important mechanism for the survival of endothelial cells.