摘要
目的探讨联合运用雷帕霉素及嘧啶亚硝脲(尼莫司汀)作用于人U87-MG胶质瘤细胞的效果及相互作用,并探讨其可能的机制。方法 MTT法检测雷帕霉素和尼莫司汀单药或联用抑制胶质瘤细胞增殖的效果并用改良寇式法计算各自的半数抑制浓度(IC50);等辐射法评价两药联合作用的相互关系;流式细胞术检测用药前后细胞周期分布和凋亡;吖啶橙及单丹(磺)酰戊二胺荧光染色检测细胞自噬;透射电镜观察细胞超微结构。结果 MTT检测显示单用或联合运用雷帕霉素及尼莫司汀均可抑制U87-MG胶质瘤细胞生长[雷帕霉素IC50为(0.726±0.063)μmol/L,尼莫司汀IC50为(26.900±2.300)μg/ml],等辐射法分析显示两药联合有协同效应;流式细胞术检测显示ACNU可诱导代表凋亡的亚G1峰出现(20.6%),而RAP可诱导细胞发生G1期阻滞(64.8%),但无明显的亚G1峰,两药联合作用后G1期细胞比例明显增多(76.0%),亚G1峰也略有增加(27.5%);吖啶橙及单丹(磺)酰戊二胺荧光染色结果表明RAP可诱导细胞发生自噬,ACNU则不明显,两药联用后自噬囊泡较单用RAP组明显增多;透射电镜可观察到两药联用后同一细胞内自噬体及凋亡同时出现。结论雷帕霉素及尼莫司汀联合作用于人U87-MG胶质瘤细胞可起协同效应,抑制U87-MG胶质瘤细胞的生长及增殖。
Objective To investigate the effect of combining use of rapamycin （RAP） and ACNU （nimuatine） on human glioma cell line U87-MG and their interaction. Methods Modified Karber’s method was used to test the lethal dose 50% （IC50） of RAP or ACNU to U87-MG cells. Then the cells were divided into control group,RAP groups （0.01,0.1,1,10 or 100 μmol/L）,ACNU groups （0.1,1,10,100 or 1 000 μg/ml）,and combining groups （ACNU at above doses combined with RAP 1/4,1/2 or 3/4 IC50）. Cell vitality of different groups was determined by MTT assay. The combined effect of RAP plus ACNU in inhibition of U87-MG cells was analyzed by isobolographic analysis. Cell cycle and apoptosis of U87-MG cells were evaluated by flow cytometry. Acridine orange and MDC staining were conducted to detect the autophagy of U87-MG cells. Ultrastructure of the cells was observed by transmission electron microscopy. Results As MTT assay showed,the tumor cell survival rate was inhibited significantly afer treatment of rapamycin alone or plus ACNU. IC50 was 0.726±0.063 μmol/L in the RAP group,and 26.9±2.3 μg/ml in the ACNU group. Isobolographic analysis demonstrated that RAP and ACNU in combination had a synergistic effect during treatment. Although ACNU significantly induced apoptosis in U87-MG cells when compared with control group,no significant apoptosis was found in RAP groups. In RAP group,cells were arrested in G1 phase. More apoptosis cells and cells arrested in G1 phase were detected in combining RAP and ACNU group. Staining of acridine orange and MDC demonstrated that RAP induced autophagy alone,which was enhanced by ACNU treatment. Autophagosomes and apoptosis were found in combining groups by transmission electron microscopy. Conclusion RAP and ACNU had synergistic interactions in suppressing the growth and proliferation of U87-MG cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2010年第14期1529-1533,共5页
Journal of Third Military Medical University
