二甲双胍对巨核细胞白血病细胞株Dami细胞增殖的抑制作用及其机制

目的：研究二甲双胍对巨核细胞白血病细胞株Dami细胞生长的影响，初步探讨二甲双胍抑制Dami细胞增殖的分子机制。方法：将培养的Dami细胞分为空白对照组,1、2、4、8、16和32 mmol?L-1二甲双胍处理组；MTT法检测不同浓度二甲双胍作用后Dami细胞增殖抑制率，流式细胞术检测细胞周期分布，免疫印迹分析Cdc2和Cyclin B1蛋白的表达以及Cdc2蛋白的磷酸化修饰。结果：MTT检测，与空白对照组比较，32 mmol/L二甲双胍处理组0、24、48、72和96 h Dami细胞增殖抑制率明显升高［（35.1±2.3）%、（49.7±5.1）%、（78.8±0.9）%、（79.1±3.0）%和（85.2±3.2）%］（P<0.01）；在二甲双胍处理72 h后，1、2、4、8、16和32 mmol/L处理组Dami细胞增殖抑制率分别为（33.8±1.3）%、（51.9±2.2）%、（59.4±1.6）%、（65.5±2.0）%、（75.5±0.9%）和（79.1±3.0）%，二甲双胍对Dami细胞的增殖抑制作用呈时间和剂量依赖性。流式细胞术检测，与空白对照组比较，1、2和4 mmol/L二甲双胍处理组G2/M期细胞比例从（26.0±0.5）%升高至（38.5±1.5）%、（48.4±1.1）%和（58.2±2.7）%，4 mmol?L-1二甲双胍处理组G2/M期细胞比例明显高于对照组（P<0.01）。免疫印迹分析，与空白对照组比较，4 mmol/L二甲双胍处理组Cdc2和Cyclin B1的表达水平明显下降，Cdc2在Try15位点磷酸化明显上调，而在Thr161位点的磷酸化明显下调。结论：二甲双胍能抑制Dami细胞增殖并诱导其发生G2/M期阻滞，其机制可能与Cdc2/Cyclin B1复合物的活化受到抑制有关。

Inhibitory effect of metformin on proliferation of megakaryocytic leukemia cell line Dami and its mechanism

Objective To study the effect of metformin on the growth of megakaryocytic leukemia cell line Dami and to explore the molecular mechanisms of the inhibitory effect of metformin on the proliferation of Dami. Methods The Dami cells were cultured and divided into control and 1,2,4,8,16and 32mmol·L-1 metformin groups.Then MTT test was performed to detect the inhitory rate of proliferation of Dami cells after treated with different concentrations of metformin.Flow cytometry was used to examine the distribution of cell cycle,and Western blotting was carried out to analyze the expressions of Cdc2and CylinB1and the phosphorylation of Cdc2. Results The MTT results showed that compared with control group,the inhibitory rates of proliferation of the Dami cells in 32 mmol·L-1 metformin groups at 0,24,48,72and 96h(35.1% ±2.3%,49.7% ±5.1%, 78.85±0.9%,79.1%±3.0%%,and 85.2% ±3.2%)were significantly increased(P<0.01),Furthermore, after metformin treatment for 72h,the inhibitory rates of proliferation of the Dami cells in 1,2,4,8,16and 32mmol·L-1 metformin groups were(33.8±0.3)%,(51.9±0.2)%,(59.4±1.6)%,(65.5±2.0)%,(75.5±0.9)%,and(79.1±3.0)%,respectively.Metformin inhibited the growth of Dami cells in a time-and dose-dependent manner.The flow cytometry results results revealed that compared with control group,the percentages of Dami cells in G2/M phase in 1,2and 4mmol·L-1 metformin groups were increased from(26.0± 0.5)%to(38.5±1.5)%,(48.4±1.1)%,and(58.2±2.7)%;there was significant difference in the percentages of Dami cells in G2/M phase between control group and 4mmol·L-1 metformin group(P<0.01). Western blotting analysis showed that compared with control group,the expressions of Cdc2and CyclinB were evidently reduced,the phosophorylation of Cdc2at Tyr15was up-regulated,and the phosphorylation at Thr161 was down-regulated.Conclusion Metformin can inhibit the growth of Dami cells and induce G2/M arrest,and its mechanism may be related to inhibiting the activation of Cdc2/CyclinB1complex.