摘要目的 探讨转录因子活化蛋白-2α(AP-2α)对香烟提取物(CSE)所致半胱氨酸天冬氨酸蛋白酶-3(Caspase 3)的活化及人脐静脉内皮细胞株ECV304凋亡的影响.方法 按照不同浓度CSE将ECV304细胞分为0.0%、2.5%、5.0%、10.0%、15.0%、20.O%CSE组;再将5.0%CSE组按照不同处理时间分为0、6、12、18、24、36和48 h组,按照不同转染腺病毒载体(Ad)分为Ad-AP-2α组、对照腺病毒载体(Ad-BgⅡ)组和对照组,按照不同转染基因及处理因素分为Ad-AP-2α+5%CSE 24 h组、Ad-BgⅡ+5%CSE 24 h组及对照+5%CSE 24 h组.每组3例.采用腺病毒介导的转染、双苯甲亚胺染核和Western blot等方法观察过表达AP-2α对CSE诱导的ECV304凋亡和Caspase 3活化的影响.采用Bartlett法进行方差齐性检验,两组间比较用t检验,多组间比较采用单因素方差分析,多个样本均数的多重比较采用LSD-t检验,多个样本率的比较采用X~2检验.结果 2.5%CSE组的细胞增殖能力(吸光度值为0.754±0.109)明显高于对照组(吸光度值为0.630±0.086),其余浓度组的细胞增殖能力逐渐下降,并呈浓度依赖的方式.随着5.0%CSE作用时间延长,细胞凋亡率从(5.0±1.0)%逐渐增到(72.6±12.1)%;转染24 h时,AP-2α的蛋白相对含量的积分吸光度值(0.882±0.014)明显高于对照组(0.635±0.005);过表达AP-2α使5.0%CSE诱导的细胞凋亡率从(7.5±0.9)%降至(0.9±0.4)%,Caspase 3蛋白相对含量从0.484±0.025降至0.300±0.020.结论 AP-2α可以抑制ECV304细胞中CSE诱导的Caspase 3活化及细胞凋亡.

abstractsObjective To investigate the effect of activator protein-2α ( AP-2α) 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). Methods 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-2α induced by CSE were tested by Western blot The effects of over-expression of AP-2a on ECV304 apoptosis and the expression of cleaved Caspase 3 were investigated by transfection, Hoechst staining and Western blot. Results 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 ( X~2 = 1773. 0, P < 0.01). The expressions of AP-2α ( 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-2a 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). Conclusion AP-2a was shown to inhibit the activation of Caspase 3 and the apoptosis of ECV304 induced by CSE.