【摘要】 目的:研究Gefitinib对人胶质瘤U251细胞的生长抑制作用及相关机制。方法:用四甲基偶氮唑盐(MTT)比色法检测不同浓度Gefitinib对U251细胞增殖活性的效应。流式细胞术(FCM)分析Gefitinib对U251细胞周期以及凋亡的影响,蛋白质印迹法检测周期、凋亡相关蛋白表达量的变化。罗丹明123/PI双染流式细胞术与吖啶橙(AO)染色荧光显微镜分析凋亡。结果:Gefitinib能显著抑制U251细胞的增殖,并呈剂量依赖关系,半数抑制浓度(IC50)为18.01μmol/L。通过流式、荧光染色以及蛋白质印迹检测分析发现,随着浓度的增加,Gefitinib能明显阻滞U251细胞于G0/G1期,主要通过降低线粒体膜电位诱导细胞凋亡,0、10、20和40μmol/L浓度处理后细胞凋亡率分别为1.28%、4.48%、77.97%和90.59%。Gefitinib对U251细胞的周期阻止与诱导凋亡主要是下调细胞周期依赖蛋白激酶CDK2、CDK4和CDK6的表达,同时上调p27Kip1的表达,引起细胞周期阻滞。上调、活化凋亡相关蛋白Bax,下调抗凋亡蛋白Bcl-2的表达,导致线粒体膜电位降低,活化Caspase-9,诱导细胞凋亡。结论:Gefitinib在15～60μmol/L浓度范围内,能明显抑制U251细胞的增殖并能通过周期阻滞诱导其凋亡,其作用呈浓度依赖关系。主要通过下调周期依赖蛋白激酶的表达阻滞U251细胞于G0/G1期,降低线粒体膜电位,活化Caspase途径诱导U251细胞凋亡。更多还原

【Abstract】 OBJECTIVE:To study the growth inhibition influence of Gefitinib on U251 gliom cells and related mechanism.METHODS:Using the method of MTT the proliferation abilities of U251 was detected in different concentrations of Gefitinib.The effect of Gefitinib on U251 cell cycle and apoptosis were analyzed by Flow Cytometry（FCM）.The expression of cell cycle and apoptosis related protein was analyzed by Western blotting.At the same time,by Flow Cytometry detecting Rhodamine 123/PI double dye and fluorescence microscopic observing acridine orange（AO） dyeing the mechanism of apoptosis was analyzed.RESULTS:Gefitinib could significantly inhibit the proliferation of U251 cell with dose-response relationship,and the half inhibition concentration（IC50） was 18.01 μmol/L.Through the analysis of Flow Cytometry,fluorescence stain and Western blotting found that Gefitinib induced G0/G1 cell cycle arrest and leaded to apoptotic cell death by decreasing the mitochondrial membrane potential.With the increasing concentration of Gefitinib（0,10,20,40 μmol/L）,it obviously induced G0/G1 cell cycle arrest.Compare to control,it mainly induced apoptosis through damaging the mitochondrial membrane potential of cell and the rate of apoptosis of each concentration were 1.28%,4.48%,77.97%,90.59%.These processes were mainly through down-regulation of cyclin-dependent kinase 2（CDK2）,CDK4,CDK6 expression,and up-regulation of p27Kip1.Up-regulating and activaing apoptosis related protein Bax and down-regulating the expression of anti-apoptotic protein Bcl-2 could decrease the mitochondrial membrane potential,and then induced apoptosis by activating and up-regulation the expression of Caspase-9.CONCLUSIONS:Between the concentration of 15 μmol/L and 60 μmol/L,Gefitinib can significantly inhibit the proliferation of U251 cell and induce apoptosis through the cycle arrest with a dose-response relationship.The Gefitinib induce G₀/G₁ cell cycle arrest mainly through down-regulation the expression of cell cycle related protein kinase,decrease the mitochondrial membrane potential,and induce apoptosis through activating the pathway of Caspase family.更多还原