哇巴因对血管内皮细胞ECV304细胞凋亡及胞内游离钙离子和活性氧浓度的影响  

Effect of ouabain on human vascular endothelial ECV304 cell apoptosis and on concentrations of intracellular free calcium ion and reactive oxygen species

作　　者：陈小义[1] 徐忠伟[1] 王娜[2] 徐瑞成[3] 

机构地区：[1]武警医学院生物学教研室,天津300162 [2]武警北京总队第一医院内三科,北京100062 [3]天津市职业和环境危害生物标志物重点实验室,天津300162

出　　处：《中国药理学与毒理学杂志》2010年第4期241-248,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：天津市自然科学基金资助项目(06YFJMJC10400)~~

摘　　要：目的研究哇巴因(毒毛花苷G)对人脐静脉血管内皮细胞ECV304凋亡的诱导作用,并探讨其可能的作用机制。方法哇巴因0.01,0.05,0.1,0.5,1和10μmol·L-1与ECV304细胞作用24,48和72h,MTT法检测细胞存活率,Hoechst33342/碘化丙锭双荧光染色法和流式细胞仪检测细胞凋亡百分率,激光共聚焦显微镜观察细胞内游离Ca2+浓度([Ca2+]i)和活性氧(ROS)浓度,逆转录PCR和Western印迹法检测胱天蛋白酶3mRNA和蛋白表达。结果哇巴因在0.01～10μmol·L-1浓度范围内与ECV304细胞分别作用24,48和72h,对细胞存活的抑制率明显增加,且呈浓度和时间依赖性,24,48和72h浓度-效应相关系数分别为0.984,0.994和0.997(P<0.05);哇巴因作用24,48和72h的IC50值分别为0.624,0.184和0.041μmol·L-1,时间-效应相关系数为0.974(P<0.05)。哇巴因0.1μmol·L-1与ECV304细胞作用24h,细胞凋亡百分率由正常对照组的(4.2±0.5)%升高到(26.0±3.2)%,作用48h,细胞凋亡率由(4.7±0.5)%升高到(36.5±5.3)%,差异有统计学意义(n=3,P<0.01);同时细胞出现染色质凝集。哇巴因0.01,0.1和0.5μmol·L-1分别与ECV304细胞作用12,24和36h,[Ca2+]i和ROS浓度呈浓度和时间依赖性增加,在哇巴因0.5μmol·L-1时[Ca2+]i和ROS浓度的时间-效应相关系数分别为0.912和0.924,作用36h时[Ca2+]i和ROS浓度的浓度-效应相关系数分别为0.889和0.907(P<0.05)。逆转录PCR和Western印迹法分析显示,哇巴因0.1和0.5μmol·L-1作用ECV304细胞24h后,胱天蛋白酶3mRNA表达增加,差异有统计学意义(P<0.05);哇巴因0.01,0.1和0.5μmol·L-1作用ECV304细胞24h,胱天蛋白酶3蛋白表达明显增加,差异有统计学意义(P<0.05)。结论哇巴因可诱导人脐静脉血管内皮细胞ECV304凋亡,其机制可能与增加[Ca2+]i和ROS浓度及胱天蛋白酶3表达有关。OBJECTIVE To study the effect of ouabain(strophanthin G) on human vascular endothelial ECV304 apoptosis and the possible mechanism.METHODS ECV304 cells were treated with ouabain 0.01,0.05,0.1,0.5,1 and 10 μmol·L-1 for 24,48 and 72 h.The survival rates of cells were detected by MTT assay;cells morphological changes were studied by Hoechst 33342 /propidium iodide staining;cell cycle distribution was detected by flow cytometry;and the concentrations of the intracellular free calcium ion([Ca2 +]i) and reactive oxygen species(ROS) in ECV304 cells were measured by the laser confocal microscope.The caspase 3 mRNA and protein expression level on ECV304 cells were detected by RT-PCR and Western blotting,respectively.RESULTS Ouabain 0.01-10 μmol·L-1 was incubated with ECV304 cells for 24,48 and 72 h.The inhibition rate of cell survival was increased in a concentration-and time-dependent manner.The coefficient of concentration-correlation was 0.984,0.994 and 0.997(P <0.05),respectively.IC50 of ouabain at 24,48 and 72 h was 0.624,0.184 and 0.041 μmol·L-1 and the coefficient of time-correlation was 0.974(P < 0.05).Compared with normal control group,the apoptosis rate of ECV304 cells treated with ouabain 0.1 μmol·L-1,from increased from(4.2 ±0.5) % to(26.0 ± 3.2) % at 24 h,and from(4.7 ±0.5) % to(36.5 ±5.3) % at 48 h,being statistically significant(n =3,P <0.01).The cells showed obviously defluxion and nuclear chromatin condensation when treated with ouabain 0.1 μmol·L-1 for 24 and 48 h.The [Ca2 +]i and ROS concentration in ECV304 cells significantly increased in a concentration-and time-dependant manner after ECV304 cells were incubated with ouabain 0.01,0.1 and 0.5 μmol·L-1 for 12,24 and 36 h,respectively.When ouabain was 0.5 μmol·L-1,the coefficient of time-correlation of [Ca2 +]i and ROS concentration were 0.912 and 0.924,respectively.When the ECV304 cells was incubated with ouabain for 36 h,the coefficient of concentration-correlation of [Ca2 +]i and ROS concentration were 0.889 and 0.907(P <0.05),respec

关 键 词：哇巴因 内皮细胞 细胞凋亡 钙 活性氧 胱天蛋白酶3 

分 类 号：R966[医药卫生—药理学]