二甲双胍诱导胃癌细胞MNK-45发生自噬的作用

目的 探讨二甲双胍诱导胃癌细胞MNK-45发生自噬的作用.方法 取对数生长期的人胃癌细胞MNK-45,接种于培养板中：采用不同浓度二甲双胍（2、4、8、16、32、64 mmol/L）分别干预24、48、72 h并作为不同浓度药物干预组,用等量DMEM培养基处理为对照组,MTT法检测肿瘤细胞的抑制率,计算二甲双胍对胃癌细胞的半数凋亡浓度（IC50）值为17 mmol/L.分别采用17 mmol/L二甲双胍（实验组）和等量DMEM培养基（对照组）处理胃癌细胞MNK-45 48 h.流式细胞仪检测两组细胞的凋亡情况;RT-PCR检测两组细胞中Bax和Bcl-2的mRNA表达水平;Western blot检测Ⅰ型微管相关蛋白轻链（LC3b Ⅰ）、Ⅱ型LC3b（LC3bⅡ）、自噬相关蛋白（beclinl）、AKT、磷酸化AKT（p-AKT）、哺乳动物雷帕霉素靶蛋白（mTOR）、磷酸化mTOR （p-mTOR）、核糖体蛋白s6激酶（P70s6k）、磷酸化P70s6k （p-P70s6k）蛋白的表达水平.计量资料采用（x）±s表示,多组间比较采用单因素方差分析,重复测量数据采用重复测量的方差分析,两组比较采用t检验.结果 MTT法检测不同浓度二甲双胍（2、4、8、16、32、64 mmol/L）分别干预胃癌细胞MNK-4524 h后细胞抑制率分别为3.0％±1.1％、8.6％±1.7％、15.9％±1.6％、26.1％±3.4％、37.5％±2.3％、49.7％±3.6％,干预48 h后细胞抑制率分别是5.2％±1.9％、10.4％±2.1％、26.9％±1.6％、49.5％±1.6％、59.1％±2.0％、82.1％±2.2％,干预72 h后细胞抑制率分别是9.5％±2.2％、l7.6％±1.4％、30.6％±2.6％、63.2％±2.6％、78.9％±1.4％、93.3％±2.7％,6组不同浓度二甲双胍干预细胞同时间点细胞抑制率比较,差异有统计学意义（F=155.174,728.229,743.826,P＜0.05）;相同浓度二甲双胍干预细胞不同时间点细胞抑制率比较,差异有统计学意义（F=39.420,58.692,166.125,30.383,117.517,311.642,P＜0.05）.流式细胞仪检测实验组和对照组胃癌细胞MNK-45的凋亡率分别为25.4％

Effects of metformin in inducing autophagy of gastric cancer MNK-45 cells

Objective To investigate the mechanisms of metformin in inducing autophagy of gastric cancer MNK-45 cells.Methods Human gastric cancer MNK-45 cells in logarithmic growth phase were incubated in the culture plates,and were divided into the intervention group [gastric cancer MNK-45 cells were intervened by metformin at different concentrations （2,4,8,16,32,64 mmol/L） for 24,48,72 hours] and the control group （gastric cancer MNK-45 cells were cultured in the DMEM medium）.The inhibition rate of gastric cancer MNK-45 cells was detected by MTT method.The IC50 value of metformin on gastric cancer MNK-45 cells was 17 mmol/L.Gastric cancer MNK-45 cells were intervened by metforrnin at 17 mmol/L for 48 hours in the experimental group.Gastric cancer MNK-45 cells in the control group were cultured in DMEM medium at 17 mmol/L for 48 hours.The apoptosis of the gastric cancer MNK-45 cells of the 2 groups were detected by flow cytometry.The mRNA expressions of Bax and Bcl-2 of the 2 groups were detected by RT-PCR.The protein expressions of type Ⅰ LC3b,type Ⅱ LC3b,beclinl,AKT,p-AKT,mTOR,p-mTOR,P70s6k,p-P70s6k of the 2 groups were detected by Western blot.The measurement data were presented as （x） s,and were analyzed using the one-way ANOVA or repeated measures ANOVA.Data of the 2 groups were compared using the t test.Results The inhibition rates of gastric cancer MNK-45 cells were 3.0% ± 1.1%,8.6% ± 1.7%,15.9% ± 1.6%,26.1% ± 3.4%,37.5% ± 2.3%,49.7%± 3.6% after intervention by metformin at concentrations of 2,4,8,16,32,64 mmol/L for 24 hours,5.2%± 1.9%,10.4%±2.1%,26.9%± 1.6%,49.5%± 1.6%,59.1%±2.0%,82.1%±2.2% after intervention by metformin at concentrations of 2,4,8,16,32,64 mmol/L for 48 hours,and 9.5% ± 2.2%,17.6% ± 1.4%,30.6% ± 2.6%,63.2% ± 2.6%,78.9% ± 1.4%,93.3% ± 2.7% after intervention by metformin at concentrations of 2,4,8,16,32,64 mmol/L for 72 hours.There were significant differences in the inhibition rates among the 6 groups at the same time points （F =155.174,728.229,743.826,P 〈 0