Li Jun-Li, Chen Ping
Department of Respiratory Medicine, Xiangya Second Hospital of Central South University, Changsha 410011, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2009 Dec;32(12):931-4.
To investigate the effect of activator protein-2alpha (AP-2alpha) on activation of cysteine proteases with aspartate specificity 3 (Caspase 3) and the apoptosis of vascular endothelial cells (ECV304) induced by cigarette smoke extract (CSE).
ECV304 were cultured in vitro, and those at the exponential growth phase were studied in experiments. The cells were cultured with 0.0%, 2.5%, 5.0%, 10.0%, 15.0%, and 20.0%CSE respectively for 24 h, and in another experiment, the cells were exposed to 5.0%CSE for 0, 6, 12, 18, 24, 36, and 48 h respectively. Then the cells were infected with different virus vectors or treated by 5%CSE alone for 24 h. Cell apoptosis, and proliferation were tested by Hoechst staining and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) respectively, while the expression of cleaved Caspase 3 and AP-2alpha induced by CSE were tested by Western blot. The effects of over-expression of AP-2alpha on ECV304 apoptosis and the expression of cleaved Caspase 3 were investigated by transfection, Hoechst staining and Western blot.
Compared with the blank control group (0.630 +/- 0.086), the proliferation was significantly increased in 2.5% CSE group (0.754 +/- 0.109), while that was decreased in the 5.0%, 10.0%, 15.0% and 20.0% CSE groups in a concentration-dependent manner. The cell apoptosis rate induced by 5.0%CSE was increased in a time-dependent manner [from (5.0 +/- 1.0)% to (72.6 +/- 12.1)%], and the differences among the groups was significant (chi(2) = 1773.0, P < 0.01). The expressions of AP-2alpha (0.882 +/- 0.014) in 24 h CSE-treatment group was higher than that of the control group (0.635 +/- 0.005, t = 5.21, P < 0.01). Over-expression of AP-2alpha inhibited the cell apoptosis [the apoptosis rate was (0.9 +/- 0.4)%, (7.5 +/- 0.9)% respectively] and the expression of cleaved Caspase 3 (the ratio were 0.300 +/- 0.020, 0.484 +/- 0.025) induced by CSE (t = 6.96, 8.75, all P < 0.01).
AP-2alpha was shown to inhibit the activation of Caspase 3 and the apoptosis of ECV304 induced by CSE.
探讨激活蛋白-2α（AP-2α）对天冬氨酸特异性半胱氨酸蛋白酶3（Caspase 3）激活及香烟烟雾提取物（CSE）诱导的血管内皮细胞（ECV304）凋亡的影响。
体外培养ECV304，选取指数生长期细胞进行实验。分别用0.0%、2.5%、5.0%、10.0%、15.0%和20.0%的CSE培养细胞24小时，在另一实验中，细胞分别暴露于5.0% CSE中0、6、12、18、24、36和48小时。然后用不同病毒载体感染细胞或单独用5% CSE处理24小时。分别采用Hoechst染色和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测细胞凋亡和增殖情况，同时采用蛋白质免疫印迹法检测CSE诱导的裂解型Caspase 3和AP-2α的表达。通过转染、Hoechst染色和蛋白质免疫印迹法研究AP-2α过表达对ECV304细胞凋亡及裂解型Caspase 3表达的影响。
与空白对照组（0.630±0.086）相比，2.5% CSE组细胞增殖显著增加（0.754±0.109），而5.0%、10.0%、15.0%和20.0% CSE组细胞增殖呈浓度依赖性降低。5.0% CSE诱导的细胞凋亡率呈时间依赖性增加[从（5.0±1.0）%增至（72.6±12.1）%]，组间差异有统计学意义（χ² = 1773.0，P < 0.01）。CSE处理24小时组AP-2α的表达（0.882±0.014）高于对照组（0.635±0.005，t = 5.21，P < 0.01）。AP-2α过表达抑制了CSE诱导的细胞凋亡[凋亡率分别为（0.9±0.4）%、（7.5±0.9）%]和裂解型Caspase 3的表达（比值分别为0.300±0.020、0.484±0.025）（t = 6.96、8.75，均P < 0.01）。
AP-2α可抑制CSE诱导的Caspase 3激活及ECV304细胞凋亡。
