celecoxib抑制肝癌细胞株SMMC-7721增殖及诱导凋亡的研究

Studies on a selective cyclooxygenase-2 inhibitor celecoxib inhibiting growth of human hepatocellular cancer SMMC-7721 cells lines and inducing apoptosis

背景与目的:肝癌发病率高,治疗效果差,通过研究选择性环氧合酶-2(COX-2)抑制剂celecoxib对人肝癌细胞株SMMC-7721增殖、凋亡的影响,为治疗肝癌提供依据。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,透射电镜观察细胞形态改变,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celecoxib在25、50、75、100μmol/L浓度下,24h抑制率分别为(15±3)%、(34.6±2.4)%、(56.8±1.0)%、(86.2±0.4)%,48h抑制率分别为(33.4±0.7)%、(66.7±1.8)%、(76.1±2.4)%、(97.3±0.8)%,(P<0.05);透射电镜观察到细胞凋亡的特征形态改变,如细胞体积缩小,胞膜皱缩,表面微绒毛消失,核固缩、异染色质沿核膜分布,有凋亡小体;流式细胞仪测定celecoxib组出现凋亡峰:24h50μmol/L组凋亡率分别为(14.6±1.1)%,100μmol/L组24、48h凋亡率分别为(40.5±2.0)%、(56.2±1.4)%,(P<0.01);DNA提取行琼脂糖凝胶电泳可见DNA的"梯状"条带,免疫组化显示经celecoxib干预后,bax基因表达增加,由阳性到强阳性,而bcl-2基因表达减少,由强阳性到弱阳性,并随时间和药物浓度变化而变化。结论:celecoxib抑制SMMC-7721细胞增殖,并呈剂量、时间依赖性,诱导SMMC-7721细胞凋亡,其机制之一可能是通过减少bcl-2基因的表达,而增加bax基因的表达,从而启动细胞凋亡。

Background and purpose：The incidence of hepatoma is high. The outcome of treatment on hepatoma is poor.So we investigated the effect and mechanism of a selective cyclooxygenase-2 inhibitor celecoxib on the proliferation and apoptosis of SMMC-7721 hepatoma cell line. Methods：MTT assay was used to study the inhibitive effect of celecoxib on the growth of SMMC-7721 hepatoma cell. The effect of celecoxib on cell cycle and apoptosis on cells was studied by flow cytometry（FCM）.Transmission electron microscopy （TEM） was used to display the morphological change of the SMMC-7721 hepatoma cell . The biochemical character of apoptosis was viewed on the agarose gel electrophoresis.The expression of bax gene and bcl-2 gene were measured by immunohistochemistry.Results：The SMMC-7721 cells were cultured in media that contained 25,50,75,100 μmol/L celecoxib,by means of MTT, the inhibition rate was（15±3）%,（34.6±2.4）%,56.8±1.0）%,（86.2±0.4）% respectively after 24 hours; but the inhibition rate was （33.4±0.7）%,（66.7±1.8）%,（76.1±2.4）%,（97.3±0.8）% respectively after 48 hours（P〈0.05）. Under the inspection of TEM,most of SMMC-7721 cells displayed the typically morphological features of apoptosis,including cell shrinkage,condensation and fragmentation of nuclear chromatine,etc. The group treated with celecoxib had an apoptotic peak in DNA histogram of FCM , but the control group had none.Treated with 50 μmol/L celecoxib, the apoptotic rate was （14.6±1.1）% after 24 hours. The apoptotic rate was （40.5±2.0）%、（56.2±1.4）%, respectively, treated with 100μmol/L celecoxib after 24 and 48 hours（P〈0.01）. DNA ladder was viewed on the Agarose gel electrophoresis. It was observed by Immunohistochemical staining that celecoxib downregulated expression of bcl-2 gene, but upregulated the expression of bax gene.Conclusions：The celecoxib inhibited the proliferation and induced the apoptosis of the SMMC-7721 cells in a dose and time dependent way. The possible mechanism may be via the reduction of the expression of bcl-2 gene, but with increase of the level of bax gene, through which induced the human hepatoma cell apoptosis by the Mitochondria/Cyt C pathway. 【Keyword】：