替莫唑胺联合二甲双胍对胶质瘤干细胞的清除作用及其机制

doi:10.13481/j.1671-587x.20150223

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (02): 321-326.doi: 10.13481/j.1671-587x.20150223

替莫唑胺联合二甲双胍对胶质瘤干细胞的清除作用及其机制

余志云¹, 李蕴潜¹, 陈勇¹, 赵丽艳², 刘焕兵¹, 王楠¹, 王斌¹, 陈云照¹, 赵刚¹

1. 吉林大学第一医院神经外科, 吉林长春 130021;
2. 吉林大学第一医院检验科, 吉林长春 130021

收稿日期:2014-12-31 出版日期:2015-03-28 发布日期:2015-04-04
通讯作者: 李蕴潜, 教授, 主任医师, 硕士研究生导师(Tel:0431-88782331, E-mail:13943188080@163.com);赵刚, 教授, 主任医师, 博士研究生导师(Tel:0431-88782331, E-mail:zhaogang@jdnwk.com) E-mail:13943188080@163.com;zhaogang@jdnwk.com
作者简介:余志云(1987-), 男, 湖北省宜昌市人, 在读医学博士, 主要从事胶质瘤基础与临床方面的研究。
基金资助:
吉林省科技厅科技发展计划项目资助课题(20140414060GH)

Elimination of temozolomide in combination with metformin on glioma stem cells and its mechanism

YU Zhiyun¹, LI Yunqian¹, CHEN Yong¹, ZHAO Liyan², LIU Huanbing¹, WANG Nan¹, WANG Bin¹, CHEN Yunzhao¹, ZHAO Gang¹

1. Department of Neurosurgery, First Hospital, Jilin University, Changchun 130021, China;
2. Department of Clinical Laboratory, First Hospital, Jilin University, Changchun 130021, China

Received:2014-12-31 Online:2015-03-28 Published:2015-04-04

摘要/Abstract

摘要：

目的:探讨替莫唑胺(TMZ)联合二甲双胍(MET)对体外胶质瘤干细胞(GSCs)的清除作用,探讨其作用机制。方法:神经干细胞培养基培养人胶质瘤U87细胞,免疫荧光法鉴定GSCs。收集GSCs,以对照液、不同浓度TMZ、MET和TMZ+MET作用细胞,分为对照组、TMZ组、MET组和TMZ+MET组。显微镜下计数各组二次神经球的数量;CCK-8法检测各组GSCs增殖抑制率;流式细胞术和Annexin-PI双染流式细胞术检测GSCs各细胞周期百分比和凋亡率。结果:与对照组比较,TMZ+MET组二次神经球数量以浓度依赖的方式减少;与TMZ和MET组比较,TMZ+EMT组二次神经球数量明显减少(P<0.01)。与对照组比较,TMZ和MET组GSCs增殖抑制率升高;TMZ+MET组GSCs增殖抑制率高于TMZ或MET组,联合指数(CI)小于1。流式细胞术,与TMZ和MET组比较,TMZ+MET组G₂/M期GSCs百分比明显升高(P<0.05);TMZ+MET组GSCs凋亡率明显高于TMZ和MET组(P<0.05)。结论:TMZ联合MET在体外能抑制GSCs的连续自我更新能力并清除GSCs,其机制可能与阻滞GSCs细胞周期进展和诱导细胞凋亡有关联。

关键词: 替莫唑胺, 二甲双胍, 胶质瘤干细胞, 自我更新

Abstract:

Objective To explore the effect of temozolomide (TMZ) in combination with metformin (MET) on the elimination of glioblastoma stem cells(GSCs) in vitro,and to clarify the mechanism.Methods The human glioma U87 cells were cultured in the neural stem cell medium,and the GSCs were identified by immunofluorescence methods;the GSCs were selected and were treated with control solution(control group), different concentrations of TMZ(TMZ group)and MET(MET group) or TMZ combined with MET(TMZ+MET group).The number of secondary neurospheres were counted under microscope, and the inhibitory rate of proliferation of GSCs was detected by CCK-8 assay;flow cytometry and Annexin Ⅴ and PI staining flow cytometry were used to detect the percentage of cell cycle and the apoptotic rate of GSCs,respectively.Results Compared with control group, the number of secondary neurospheres in TMZ+MET group was significantly decreased in a concentration-dependent manner;the number of secondary neurosphere in TMZ+MET group was significantly lower than those in TMZ group and MET group(P<0.01).Compared with control group, the inhibitory rates of the proliferation of GSCs in TMZ and MET groups were increased,the inhibitory rate of proliferation of GSCs in TMZ+MET group was higher than those in TMZ group and MET group(P<0.05),and the combination index(CI)<1.The flow cytometry results displayed that the percentage of the cells at G₂/M phase in TMZ+MET group was significantly higher than those in TMZ group and MET group(P<0.05),and the apoptotic rate of GSCs in TMZ+MET group was significantly higher than those in TMZ group and MET group (P<0.05).Conclusion The combination of TMZ and MET can inhibit the serial self-renewal capability of GSCs as well as eliminate GSCs,and its mechanism may be related to the inhibition of the cell cycle progression of GSCs and induction of apoptosis.

Key words: temozolomide, metformin, glioblastoma stem cells, self-renewal

中图分类号:

R730.264

余志云, 李蕴潜, 陈勇, 赵丽艳, 刘焕兵, 王楠, 王斌, 陈云照, 赵刚. 替莫唑胺联合二甲双胍对胶质瘤干细胞的清除作用及其机制[J]. 吉林大学学报(医学版), 2015, 41(02): 321-326.

YU Zhiyun, LI Yunqian, CHEN Yong, ZHAO Liyan, LIU Huanbing, WANG Nan, WANG Bin, CHEN Yunzhao, ZHAO Gang. Elimination of temozolomide in combination with metformin on glioma stem cells and its mechanism[J]. Journal of Jilin University Medicine Edition, 2015, 41(02): 321-326.

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替莫唑胺联合二甲双胍对胶质瘤干细胞的清除作用及其机制

Elimination of temozolomide in combination with metformin on glioma stem cells and its mechanism

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