二甲双胍对ALDH+胃癌干细胞的抑制作用及其机制

摘要

目的 探讨二甲双胍(Met)对乙醛脱氢酶(ALDH)阳性胃癌干细胞的抑制作用及其可能机制.方法 选择人胃癌细胞系MKN45作为亲本细胞,采用流式细胞仪分选出ALDH+和ALDH-细胞,进行白我更新能力、分化能力、裸鼠移植瘤实验,鉴定其是否为胃癌干细胞及是否具备肿瘤干细胞特性.实验设置lmmol/L Met组、5mmol/LMet组和MKN45细胞组,分别作用48h后,检测各组ALDH+细胞的比例;RT-PCR实验检测各组Oct4、Sox2、AKT基因表达量的变化.结果 细胞鉴定实验结果显示,ALDH+细胞的自我更新能力、分化能力、致瘤能力均高于ALDH细胞,表明ALDH+细胞具有肿瘤干细胞特性.实验结果显示,MKN45细胞组、1mmol/L Met组、5mmol/L Met组ALDH+细胞比例分别为36.5％±5.4％、15.6％±1.9％和7.6％±1.6％,三组差异有统计学意义(P＜o.01).RT-PCR结果显示,Met处理后Sox2和AKT基因的表达量均下降,且随Met浓度升高下降更为明显;Oct4基因在1mmol/L Met组的表达量高于MKN45细胞组,5mmol/L Met组的表达量低于MKN45细胞组.结论 Met能抑制ALDH+胃癌干细胞生长,抑制AKT基因的表达,可作为临床治疗胃癌的靶点药物.%Objective To investigate the inhibitory effect of metformin (Met) on ALDH positive (ALDH+) gastric cancer stem cells and its mechanism.Methods ALDH+ and ALDH cells were isolated from human gastric cancer cell line MKN45 by flow cytometry.The characteristics of cancer stem cells of ALDH+ cells was verified by self-renewing ability,differentiation capacity experiments and tumorigenicity in nude mice.1mmol/L Met group,5mmol/L Met group and control group (MKN45 cell) were set up.After being acted on MKN45 cell for 48h,the proportion ofALDH+ cells in each group was detected by flow cytometry.RT-PCR test was performed to detect the expression of Oct4,Sox2 and AKT genes in the 3 groups.Results The cell identification showed that the self-renewal ability,differentiation capacity and tumorigenicity of ALDH+ cells were higher than that of ALDH-cells.Drug experiments indicated that the proportion ofALDH+ cells in control group,lmmol/L Met group and 5mmol/L Met group were 36.5％ ± 5.4％,15.6％ ± 1.9％ and 7.6％ ± 1.6％,respectively.The difference between the 3 groups was statistically significant (P＜0.01).RT-PCR revealed that the expressions of Sox2 and AKT genes decreased after Met treatment,and decreased with the increase of Met concentration.The expression of Oct4 gene was higher in 1 mmol/L Met group than in control group,and was lower in 5mmol/L Met group than in control group.Conclusion Met may inhibit the growth ofALDH+ gastric cancer stem cells and the expression ofAKT gene,and can be used as a target drug for the clinical treatment of gastric cancer.