表阿霉素与替莫唑胺联合应用对C6脑胶质瘤细胞增殖及凋亡作用  

Effect of the Combined Use of Epirubicin and Temozolomide in the Proliferation and Apoptosis of C6 Glioma Cells

作　　者：轩亚茹 应雪[1] 张春春[1] 王亚华[1] 闫荷露 李霞[1] 

机构地区：[1]石河子大学药学院特种植物药资源教育部重点实验室,新疆石河子市832000

出　　处：《中国全科医学》2016年第8期925-930,共6页Chinese General Practice

基　　金：国家自然科学基金资助项目(81160396;81460540);石河子大学杰出青年科技人才培育计划项目(2012ZRKXJQ07);石河子大学大学生创新创业训练计划项目(SRP2015256);新疆生产建设兵团社会发展科技攻关与成果转化计划项目(2015AD007);国家级大学生创新创业训练计划项目(201510759066)

摘　　要：目的探讨表阿霉素(EPI)与替莫唑胺(TMZ)联合用药对C6脑胶质瘤细胞和细胞肿瘤球的增殖抑制及诱导凋亡作用。方法培养C6脑胶质瘤细胞,取对数生长期细胞活性>95%的细胞进行实验。取C6脑胶质瘤细胞接种于96孔细胞培养板中培养,24 h后加入各药液10μl至细胞培养孔中,细胞培养孔中EPI浓度梯度为0.05、0.10、0.50、1.00、5.00、10.00μmol/L,TMZ的浓度梯度为0.1、0.5、1.0、5.0、10.0、20.0μmol/L,继续培养24h和48 h。采用酶标仪在540 nm波长处测定每孔吸光度(OD)值,计算各孔C6脑胶质瘤细胞的抑制率。取C6脑胶质瘤细胞接种于6孔板中,加入EPI、TMZ、EPI+TMZ(EPI终浓度为0.50、1.00、5.00μmol/L,TMZ终浓度为1.0、5.0、10.0μmol/L),继续培养24 h和48 h,经流式细胞仪检测EPI和TMZ联合对C6脑胶质瘤细胞的凋亡作用。培养C6脑胶质瘤细胞肿瘤球,加入EPI、TMZ、EPI+TMZ,EPI终浓度为5.00μmol/L,TMZ的终浓度为10.0μmol/L,分别于给药后0、1、3、7 d于倒置显微镜下观察肿瘤球的形态、球体是否有裂解、球核是否有坏死等现象。结果不同浓度的各药物在24、48 h对C6脑胶质瘤细胞增殖的抑制率比较,差异有统计学意义(P<0.05);各浓度EPI+TMZ联合用药对C6脑胶质瘤细胞增殖的抑制率均高于相同EPI或TMZ浓度下单独用药,差异均有统计学意义(P<0.05)。培养至24 h时,1.00μmol/L EPI与5.0μmol/L TMZ联合用药对C6脑胶质瘤细胞的增殖抑制率呈协同作用;培养至48 h时,0.50μmol/L EPI与1.0μmol/L TMZ联合用药对C6脑胶质瘤细胞的增殖抑制率呈协同作用。培养至24、48 h时,阴性对照、各浓度的EPI、TMZ及EPI+TMZ对C6脑胶质瘤细胞凋亡率的比较,差异有统计学意义(P<0.05)。经EPI、TMZ及其联合用药后肿瘤球体积明显受到抑制,表现为体积缩小,表面细胞裂解;联合用药较单独用药后肿瘤球球体致密性减小,裂解程度增大。结论 EPI和TMZ联合用药对C6胶质瘤细胞具有协同抑Objective To investigate the effect of the combined used of epirubicin（ EPI） and temozolomide（ TMZ）on the proliferation and apoptosis of C6 glioma cells and C6 glioma spheroids. Methods To carry out the experiments,we used C6 glioma cells which were in the logarithmic phase and their cell activity was more than 95%. These C6 glioma cells were seeded into 96- well culture plates for 24 h. Then EPI and TMZ were severally added into 96- well culture plates with 10μl each and the concentration gradients were 0. 05,0. 10,0. 50,1. 00,5. 00,10. 00 μmol / L and 0. 1,0. 5,1. 0,5. 0,10. 0,20. 0 μmol /L,respectively. 24 h and 48 h later,the absorbance was read on a microplate reader at 540 nm. The inhibition rate of C6 gliomacells in each hole was calculated after the above process. C6 glioma cells were seeded into 6- well culture plates and then EPI,TMZ and EPI ＋ TMZ were added and the final concentrations of EPI were 0. 50,1. 00,5. 00 μmol / L and those of TMZ were1. 0,5. 0,10. 0 μmol / L,respectively. After continuous culture for 24 h and 48 h,the apoptosis effect of EPI combined with TMZ was determined by flow cytometry. C6 glioma spheroids in vitro were developed and then EPI,TMZ,EPI ＋ TMZ were added and the final concentrations of EPI and TMZ were 5. 00 μmol / L and 10. 0 μmol / L respectively. On the 0,1,3,7 d after adding the drugs,we used the inverted microscope to observe the morphologic changes of C6 glioma spheroids to see if the spheroids produced splitting decomposition and the spheroids nuclear had necrosis. Results The inhibition rates of C6 glioma cells were significantly different among the drugs of different concentrations at 24 h and 48 h（ P〈0. 05）; the inhibition rates of EPI ＋ TMZ were significantly higher than EPI or TMZ alone under the same concentration（ P〈0. 05）. The inhibition rates of C6 glioma cells showed synergy after adding 1. 00 μmol / L EPI combined with 5. 0 μmol / L TMZ at 24 h and 0. 50 μmol / L EPI combined with 1. 0 μmol / L TMZ at 48 h. At 24 h and

关 键 词：神经胶质瘤 表柔比星 替莫唑胺 细胞增殖 细胞凋亡 

分 类 号：R730.264[医药卫生—肿瘤]