二甲双胍对小鼠睾丸间质细胞的体外抑制作用及其机制研究

Inhibitory effect of metformin on mouse testicular Leydig cells in vitro and underlying mechanisms

目的探讨二甲双胍在小鼠睾丸发育中的作用及其分子机制。方法不同浓度二甲双胍作用于小鼠睾丸间质细胞(TM3),采用MTT法、Annexin V-FITC/PI法及流式细胞术检测二甲双胍对TM3细胞增殖、凋亡及周期的影响。采用real-time PCR检测细胞周期关键调节蛋白cyclin B1和cyclin D1的变化,Western Blot分析二甲双胍抑制TM3细胞增殖中AMPK蛋白的变化。结果 MTT结果显示二甲双胍可显著抑制TM3细胞的体外增殖能力;Annexin V-FITC/PI染色法显示二甲双胍未诱导该细胞发生凋亡;流式细胞术检测发现二甲双胍可诱导TM3细胞周期阻滞在G2/M期;real-time PCR显示二甲双胍可下调cyclinB1和cyclin D1 m RNA的表达;Western Blot显示二甲双胍显著增加TM3细胞中AMPK在Thr172位点的磷酸化水平。结论二甲双胍显著抑制TM3细胞的体外增殖并通过诱导TM3细胞周期阻滞在G2/M期,激活AMPK信号通路,发挥其对TM3细胞的体外抑制作用。

Objective To investigate the role and molecular nmchanism of metfornfin in mouse testicular development. Methods The effects of metfornfin on the proliferation, apoptosis and cell cycle of TM3 cells were detected by MTT assay, Annexin V-FITC/PI staining and flow- cytometly. Real-time PCR and Western blot were used to analyze the expression of cell cycle protein and AMPK （phospho172） to evaluate the effect of metfornfin on TM3 cells. Results MTT assay showed that metformin signifcantly inhibited the proliferation of TM3 cells in vitro. Annexin V-FITC/PI staining showed that metformin did not induce apoptosis of cells. Flow- cytometlT showed that metformin could induce TM3 cell cycle arrest at G2/M phase. Real-time PCR showed that the expression of cyclin B1 and cyclin D1 were down-regulated in metformin treated TM3 cells. Western blot showed that the phosphoryla- tion of AMPK at Thr172 was significantly increased in metformin treated TM3 cells. Conclusion Metfomfin significantly inhibits the proliferation of TM3 cells in vitro and induces the TM3 cell cycle arrest in G2/M phase, ac- tivating AMPK signal pathway, and exerting inhibitory effect on TM3 cells in vitro.