【摘要】 目的:探讨二甲双胍通过抑制Wnt/β-catenin信号通路对胃癌干细胞增殖和凋亡的影响。方法:常规培养胃癌MKN-45细胞,并分选其干细胞,采用免疫荧光法观察分选前后MKN-45细胞中CD44~+表达情况,通过流式细胞术检测分选前、后MKN-45细胞中CD44~+比例,并通过免疫印迹法检测悬浮MKN-45肿瘤细胞球和贴壁细胞中性别决定区Y框蛋白2(SOX2)、八聚体结合转录因子4(OCT4)、同源域蛋白(Nanog)的蛋白表达。采用不同剂量二甲双胍(0,1,5,10 mmol/L)处理MKN-45干细胞24～72 h, RT-qPCR检测MKN-45干细胞Wnt/β-catenin信号通路基因表达情况,CCK-8法检测各组细胞增殖活力,流式细胞术检测各组细胞凋亡情况。结果:与胃癌干细胞分选前相比,分选后CD44~+比例在MKN-45细胞中由5.70%富集至89.32%;悬浮MKN-45肿瘤细胞球中肿瘤干细胞标志物SOX2、OCT4、Nanog蛋白高于贴壁细胞(P<0.05),提示胃癌干细胞分离成功。随着二甲双胍剂量的增加及处理时间的延长,MKN-45干细胞增殖活性、β-连环蛋白(β-catenin)呈下调趋势,其细胞凋亡率、糖原合成酶激酶-3β(GSK-3β)呈上调趋势(P<0.05)。结论:随着二甲双胍浓度增加及作用时间延长,胃癌干细胞增殖活性增强,凋亡率降低,其作用原因可能与抑制Wnt/β-catenin信号通路的激活有关。更多还原

【Abstract】 Objective:To investigate the effect of metformin on the proliferation and apoptosis of gastric cancer stem cells by inhibiting Wnt/β-catenin signaling pathway.Methods:Routine culture of gastric cancer MKN-45 cells was performed, and the stem cells were separated.The expression of CD44~+ in MKN-45 cells before and after separation was observed by immunofluorescence method, the proportion of CD44~+ in MKN-45 cells before and after separation was detected by flow cytometry, and the protein expressions of SOX2,OCT4 and Nanog in suspended MKN-45 tumor cell spheres and adherent cells were detected by Western blotting.MKN-45 stem cells were treated with different doses of metformin(0,1,5,10 mmol/L) for 24～72 h.Gene expression in the wnt/β-catenin signaling pathway of MKN-45 stem cells was detected by RT-qPCR.Cell proliferation activity in each group was detected by CCK-8 assay, and cell apoptosis was detected by flow cytometry.Results:The proportion of CD44~+ in MKN-45 cells after sorting was enriched from 5.70% to 89.32% compared with that before sorting, and SOX2,OCT4 and Nanog proteins in suspended MKN-45 tumor cells were significantly higher than those in adherant cells(P<0.05),indicating that gastric cancer stem cells were successfully isolated from gastric cancer cell line MKN-45.With the increase of metformin dose and prolonging of treatment time, the proliferation activity and β-catenin of MKN-45 stem cells were down-regulated, while the apoptosis rate and GSK-3β were up-regulated(P<0.05).Conclusion:With the increase of metformin concentration and the prolongation of action time, the proliferation activity of gastric cancer stem cells is enhanced and the apoptosis rate decreased, which is related to the inhibition of the activation of Wnt/β-catenin signal pathway.更多还原