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目的:探讨氧化应激诱导人脐静脉内皮细胞凋亡与细胞存活信号通路PI3k/Akt-Bcl-2的关系。方法:不同浓度(0、50、100、200、400、800μmol/L)第三丁基过氧化氢(t-BHP)处理人脐静脉内皮细胞(HUVEC)1～4 h,MTT法检测内皮细胞生存率,Hoechst/PI双染色荧光显微镜观察细胞凋亡率。再选用最佳t-BHP浓度(100μmol/L)干预HUVEC不同时间(0、0.5、1、2、4、6 h),Western blot测Akt、p-Akt、Bcl-2以及caspase-3在各时间点的表达情况。结果:MTT显示随着t-BHP作用浓度的增加和时间的延长,HUVEC生存率逐渐下降,并呈现一定的量效和时效关系;Hoechst/PI染色荧光显微镜观察证实t-BHP能够诱导HUVEC发生凋亡。t-BHP浓度在50～200μmol/L时,随着其作用浓度的增加细胞凋亡率明显增加(8.2%±0.3%、37.2%±0.3%、55.6%±0.2%,P<0.05),大于200μmol/L凋亡率反而下降(44.9%±0.2%、36.8%±0.4%,P<0.05);Western blot结果显示caspase-3的活化片段在4 h表达量最高。p-Akt在30 min时表达达高峰,随后降低。Bcl-2表达呈现递减的趋势。结论:t-BHP诱导caspase依赖的HUVEC凋亡,这一病理过程与p-Akt的迅速减少和Bcl-2的减少有关。
AIM: To investigate the relationship between oxidative stress-induced apoptosis of human umbilical vein endothelial cells and cell survival signaling pathway PI3k / Akt-Bcl-2. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of t-BHP (0,50,100,200,400,800μmol / L) for 1 to 4 h. The levels of endothelial cells Survival rate, Hoechst / PI double staining fluorescence microscope to observe the rate of apoptosis. The optimal concentration of t-BHP (100μmol / L) was chosen to interfere with HUVEC at different time points (0, 0.5, 1, 2, 4 and 6 h). The expressions of Akt, p-Akt, Bcl-2 and caspase- Time point of expression. Results: The MTT showed that the survival rate of HUVEC decreased gradually with the increase of the concentration of t-BHP and the prolongation of time, and showed some dose-effect and time-effect relationship. Hoechst / PI staining confirmed that t-BHP could induce the apoptosis of HUVEC Death. When the concentration of t-BHP was in the range of 50 ~ 200μmol / L, the apoptosis rate was significantly increased with the concentration increasing (8.2% ± 0.3%, 37.2% ± 0.3%, 55.6% ± 0.2%, P <0.05) The apoptosis rate of 200μmol / L was decreased (44.9% ± 0.2%, 36.8% ± 0.4%, P <0.05). Western blot results showed that the expression of caspase-3 was highest at 4 h. The expression of p-Akt peaked at 30 min, then decreased. Bcl-2 expression showed a decreasing trend. Conclusion: t-BHP induces caspase-dependent HUVEC apoptosis. This pathological process is related to the rapid decrease of p-Akt and the decrease of Bcl-2.