吉西他滨诱导神经胶质瘤细胞凋亡及其对促凋亡因子Bax/Bak的影响  

Gemcitabine induced glioma cell apoptosis and its effect on proapoptotic protein Bax/Bak

作　　者：王华[1] 刘艳飞[2] 

机构地区：[1]南华大学附属第一医院药剂科,湖南衡阳421001 [2]中南大学化学化工学院,湖南长沙410083

出　　处：《中国医院药学杂志》2015年第23期2092-2096,共5页Chinese Journal of Hospital Pharmacy

基　　金：湖南省自然科学省市联合基金(编号:12JJ9046)

摘　　要：目的:研究抗癌药物吉西他滨(Gemcitabine,Gem)对人神经胶质瘤U87细胞的凋亡以及其对促凋亡因子Bax/Bak的影响。方法:MTT法检测0(对照组)、0.5,1,5,10μmol·L-1的Gem对U87细胞增殖的影响以及5μmol·L-1 Gem处理U87细胞12,24,36,48 h后的细胞活性。细胞核特异性染料DAPI核染后共聚焦显微镜下观察Gem处理的U87细胞核型的变化。流式细胞仪结合染色和免疫荧光技术检测细胞的凋亡情况、细胞周期分布以及Bax/Bak活化情况。Western blot检测shRNA基因沉默前后Gem引起的细胞内Bax/Bak蛋白的表达情况。MTT法检测沉默Bax/Bak后Gem对U87细胞活力的影响。结果:Gem对U87细胞活性的影响具有显著的浓度和时间依赖性。5μmol·L-1的Gem处理细胞12,24,36,48 h后细胞活力分别(79±3.2)%、(41.2±2.6)%、(27.9±3.1)%、(20.4±1.7)%。Gem处理U87细胞后细胞核明显固缩并形成凋亡小体。Gem引起细胞凋亡,5μmol·L-1 Gem处理的细胞凋亡率为(45.9±2.6)%,显著高于对照组的(3.1±1.8)%。Gem能够显著将细胞阻滞在Sub-G1和S期(P<0.01),并明显引起细胞内Bak的活化而没有引起Bax的活化(P<0.01)。与对照组相比,5μmol·L-1的Gem处理U87细胞后,沉默Bak基因很好的抑制了Bak蛋白的表达而没有抑制Bax蛋白的表达。最后发现,沉默Bak明显抑制了Gem对U87细胞的毒性(P<0.01),而沉默Bax相比Gem单独用药组没有明显变化。结论:Gem能够抑制U87细胞的增殖并引起细胞凋亡,在凋亡通路中引起细胞Sub-G1和S期的阻滞,并且导致细胞内Bak而非Bax的活化而促进凋亡。OBJECTIVE To investigate effects of clinical anticancer drug gemcitabine on glioma cell U87 apoptosis and on proapoptotic protein Bax/Bak.METHODS U87 cells were treated by 0（control group）,0.5,1,5,10μmol·L-1 gemcitabine for 24 h and 5μmol·L-1 for 12,24,36,48 h.MTT assay was used to test cell viability.U87 cells were stained by DAPI to observe nucleus change under confocal microscopy.Cell apoptosis and apoptosis ratio,cell cycle and Bax/Bak activation in cells were detected by flow cytometry.Western blot was used to detect Bax/Bak protein expression before and after silencing Bax/Bak gene by shRNA.Cell viability of U87 after silencing Bax/Bak gene was tested by MTT assay.RESULTS Effects of gemcitabine on U87 cell viability were dose and time independent（P〈0.01）.U87 cells treated with5μmol·L-1 gemcitabine after12,24,36,48 h had（79±3.2）%,（41.2±2.6）%,（27.9±3.1）% and（20.4±1.7）%cell viability,respectively.Nuclei of U87 cells became shrunk and showed apoptotic bodies.5μM gemcitabine could induce U87 cell apoptosis by（45.9±2.6）%which as significant higher than that in control group（3.1±1.8）%,Sub-G1 and S arrest（P〈0.01）,and activation of Bak rather than Bax（P〈0.01）.Compared with control group,Bak rather than Bax protein expression in U87 was investigated after gemcitabine administration.After silencing Bax/Bak genes,gemcitabine could only inhibit cell viability of Bak rather than Bax silencing group.CONCLUSION Gemcitabine can inhibit U87 cell viability and induce cell apoptosis.On apoptosis pathway,gemcitabine can result in Sub-G1 and S arrest as well as Bak rather than Bax protein activation.

关 键 词：吉西他滨 U87细胞 凋亡 western BLOT Bax/Bak 沉默RNA 

分 类 号：R979.1[医药卫生—药品]