环氧合酶-2对香烟提取物诱导的内皮细胞凋亡的作用

目的 探讨环氧合酶-2在香烟提取物(CSE)所诱导的内皮细胞凋亡中的作用.方法 体外培养人血管内皮细胞株ECV304,按3个步骤完成实验:(1)用0.0%、0.5%、1.0%和5.0%CSE分别F预ECV-304细胞12 h;(2)用5.0%CSE分别干预ECV304细胞0、3、6、9、12和24 h;(3)用5.0% CSE 及0.0、2.5、5.0、10.0、20.0、50.0μmol/L选择性环氧合酶-2抑制剂赛来昔布联合干预ECV304细胞9 h.采用Hoechst染色法和流式细胞术检测内皮细胞凋亡,免疫细胞化学和Western blot 法检测环氧合酶-2蛋白的表达.采用Bartlett法进行方差齐性检验,多组间均数比较采用单因素方差分析和LSD-t检验.结果 随着CSE干预浓度的升高,内皮细胞凋亡率和环氧合酶-2蛋白表达水平均逐渐升高,5.0%CSE干预后内皮细胞凋亡率最高(5.40±0.39)%],环氧合酶-2蛋白表达也最高(206.1±15.5),差异均有统计学意义(F值分别为90.03和159.94,均P<0.05).5.0%CSE干预内皮细胞3 h,环氧合酶-2蛋白的表达开始增加,9 h达峰值,12 h开始减弱,24 h恢复止常.随着干预时间的延长,内皮细胞凋亡率逐渐升高,5.0%CSE干预24 h内皮细胞凋亡率最高(8.87±0.41)%],差异均有统汁学意义(F=155.65,均P<0.05).5.0%CSE与不同浓度的选择件环氧合酶-2抑制剂赛来昔布共同作用于内皮细胞9 h后,随着赛来昔布干预浓度升高,环氧合酶-2的表达逐渐减弱;内皮细胞凋亡率首先出现小幅的下降,随后明显增加,以5.0%CSE+50μmol/L赛来昔布组的内皮细胞凋亡率最高(32.60±5.51)%],差异均有统计学意义(F=81.28,均P<0.05).结论 CSE能够诱导内皮细胞凋亡和环氧合酶-2蛋白表达.选择性环氧合酶-2抑制剂赛来昔布能够抑制CSE诱导内皮细胞表达环氧合酶-2蛋白,并促进内皮细胞凋亡,甚至诱发其死亡.环氧合酶-2有助于缓解CSE诱导的内皮细胞凋亡,对内皮细胞有一定程度的保护作用.

Effect of cyclooxygenase-2 on vascular endothelial cell apoptosis induced by cigarette smoke extract

Objective To explore the effect of eyelooxygenase-2 on vascular endothelial cell apoptosis induced by cigarette smoke extract. Methods Human vascular endothelial cells(ECV-304)were cultured in vitro.and those at the exponential growth phase were studied in experiments.The experiment was completed through 3 steps:(1) ECV-304 cells were cultured with 0.0%,0.5%,1.0% and 5.0% CSE for 12 h.(2)ECV-304 cells were exposed to 5.0%CSE for 0,3,6,9,12 and 24 h.(3)Endothelial cells were treated by 5% CSE,together with different concentrations of selective COX-2 jnhibitor celecoxib(0.0,2.5,5.0,10.0,20.0,50.0μmol/L concentrations)for 9 h.The cell apoptosis rate was tested by Hoechst staining and flow cytometry methods,and the expression of COX-2 protein by immunocytochemistry and Western blotting.Results CSE induced ECV-304 cell apoptosis and COX-2 expression in a dose-dependent manner.The apoptosis rate of ECV-304 cells with 5.0% CSE was the highest (5.40±0.39)%.CSE-induced COX-2 expression reached the highest level with 5.0%CSE(206.1±15.5).the difierences being significant (F=90.03,159.94,all P<0.05).Furthemmre CSE induced both apoptosis rate and COX-2 expression time-dependently,with the apoptosis rate achieving the peak after 24 h(8.87±0.41)%,while COX-2 expression reached the highest level at 9 h.The selective COX-2 inhibitor celecoxib inhibited COX-2 protein expression partially and augmented cell apoptosis induced by CSE.Conclusions CSE induces endothelial cell apoptosis and increases the expression of COX-2 protein in vascular endothelial cells.Celecoxib.the selective COX-2 inhibitor,reduces the expression of COX-2 protein and promotes cell apoptosis induced by CSE in vascular endothelial cells.COX-2 may play an important role in protecting development of CSE-associated apoptosis of endothelial cells.