摘要
目的:研究特异性环氧化酶-2(COX-2)抑制剂塞来昔布对人胃癌细胞株MKN-45是否有抑制增殖、诱导凋亡作用并初步探讨其作用机制。方法:采用四氮唑盐比色法(MTT法)观察细胞增殖活力改变;流式细胞仪检测细胞凋亡率及细胞周期变化;透射电镜观察细胞超微结构的改变;DNA提取行琼脂糖凝胶电泳观察DNA的"梯状"条带;免疫组化观察bcl-2基因和bax基因表达情况。结果:MTT法显示随着塞来昔布浓度和作用时间增加,细胞抑制率上升。人胃癌细胞株MKN-45在25、50、75、100μmol/L塞来昔布浓度下,24小时细胞抑制率分别为(16.39±1.430%、(23.55±0.11)%、(62.78±1.42)%、(87.53±0.27)%,48小时细胞抑制率分别为(30.40±0.68)%、(46.18±1.80)%、(89.77±0.21)%、(97.94±0.18)%,(P<0.05);流式细胞仪测定塞来昔布组出现凋亡峰,50、100μmol/ml浓度的塞来昔布干预24小时后,人胃癌细胞株MKN-45凋亡率分别为(25.5±2.66)%、(57.23±2.19)%;透射电镜观察显示塞来昔布组细胞呈凋亡表现,核固缩、碎裂,染色质浓缩,边集;塞来昔布(50μmol/L、100μmol/L)组培养48小时后DNA琼脂糖凝胶电泳可见典型的梯状条带,而对照组细胞DNA未见梯状条带;免疫细胞化学法显示经塞来昔布干预后,凋亡蛋白Bax表达增加,而Bcl-2表达减少,并随时间和药物浓度变化而明显。结论:塞来昔布呈剂量、时间依赖性地抑制MKN-45细胞增殖,促进MKN-45细胞凋亡。其机制之一可能是通过减少Bcl-2的表达、增加Bax的表达,从而启动细胞凋亡途径。
Objective:To investigate the effect and mechanism of celecoxib,a selective cyclooxygenase-2 inhibitor,on inhibiting the proliferation and inducing apoptosis in gastric cancer cell line MKN-45.Methods:MTT assay was used to study the inhibitive effect of celecoxib on the growth of gastric cancer cell.The effect of celecoxib on cell cycle changes and apoptosis of cells was studied by flow cytometry(FCM).The expression of Bax and Bcl-2 proteins were measured by immunohistochemistry.Ultramicrostructure was observed through transmission electron microscope(TEM).DNA gel electrophoresis was performed for assessing the effect of celecoxib on DNA apoptotic ladder.Results:It was observed that after treatment with celecoxib,the proliferations of MKN-45 cell were inhibited in a dose-and time-dependent manner.After being exposed to various concentrations of celecoxib(25,50,75 and 100μmol/L) for 24h,the inhibitive rates on MKN-45 were(16.39±1.43)%,(23.55±0.11)%,(62.78±1.42)%,(87.53±0.27)% respectively,and for 48h the inhibitive rates increased to(30.40±0.68)%,(46.18±1.80)%,(89.77±0.21)%,(97.94±0.18)% in MKN-45 respectively(P〈0.05).The apoptotic rates were(25.5±2.66)% and(57.23±2.19)% in MKN-45 after treatment with celecoxib(50,100μmol/L) for 24h.Correspondingly,the apoptotic peaks were observed by FCM at these two concentrations.In addition,it was observed that celecoxib could decrease the expression of Bcl-2,but promoted the expression of Bax protein.The apoptotic morphology,such as karyopycnosis and apoptotic body,could be observed by TEM in celecoxib groups.The 50μmol/L and 100μmol/L celecoxib groups produced characteristic DNA ladder on DNA gel,but not in the control.Conclusion:Celecoxib could inhibit the proliferation and induce the apoptosis of the MKN-45 cells in a dose-and time-dependent way.The possible mechanism may be celecoxib could reduce the expression of Bcl-2 and increase the expression of Bax.
出处
《现代肿瘤医学》
CAS
2009年第3期416-420,共5页
Journal of Modern Oncology
基金
江苏省肿瘤医院青年科技基金项目(编号:ZQ2000419)
关键词
塞来昔布
胃癌细胞株
细胞增殖
凋亡
celecoxib
human gastric cancer cell line
proliferation
apoptosis