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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。
Objective: To investigate the scavenging effect of temozolomide (TMZ) combined with metformin (MET) on glioma stem cells (GSCs) in vitro and its mechanism of action. Methods: Human glioma U87 cells were cultured in neural stem cell culture medium and GSCs were identified by immunofluorescence. The GSCs were collected and divided into control group, TMZ group, MET group and TMZ + MET group with control liquid, different concentrations of TMZ, MET and TMZ + MET. The number of secondary neurospheres in each group was counted under microscope. The proliferation inhibition rate of GSCs in each group was measured by CCK-8 method. The percentage of cell cycle and the percentage of apoptotic cells were detected by flow cytometry and Annexin-PI double staining flow cytometry. Results: Compared with control group, the number of secondary neurospheres in TMZ + MET group decreased in a dose-dependent manner. Compared with TMZ and MET groups, the number of secondary neurospheres in TMZ + EMT group decreased significantly (P <0.01). Compared with the control group, the proliferation inhibition rate of GSCs in TMZ and MET groups increased. The proliferation inhibition rate of TMZ + MET group was higher than TMZ or MET group, and the combined index (CI) was less than 1. Flow cytometry showed that the percentage of GSCs in G2 / M phase in TMZ + MET group was significantly higher than that in TMZ and MET groups (P <0.05), and the apoptotic rate of TMZ + MET group was significantly higher than that in TMZ and MET groups (P <0.05) ). CONCLUSION: TMZ combined with MET can inhibit the continuous self-renewal ability of GSCs and clear GSCs in vitro. The mechanism may be related to arresting cell cycle progression and inducing apoptosis of GSCs.