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目的:探讨二甲双胍对核糖体40S小亚基S6K蛋白激酶(P70S6k)及胰岛素受体底物-1(IRS-1)蛋白及其ser307位点磷酸化表达的影响。方法:利用0.1 mmol/L二甲双胍体外连续作用多囊卵巢综合症(PCOS)患者黄素化颗粒细胞24 h为实验组,设未加二甲双胍培养的颗粒细胞为对照组。通过RT-PCR和Real-time PCR检测P70S6k和IRS-1的表达,细胞免疫荧光化学和Western blotting的方法检测P70S6k、p-thr389-P70S6k、IRS-1及p-ser307-IRS-1蛋白的表达。结果:Real-time PCR结果显示实验组P70S6k和IRS-1m RNA水平显著低于对照组,差异有统计学意义(P<0.05)。细胞免疫荧光化学检测IRS-1和p-ser307-IRS-1蛋白在颗粒细胞胞质表达,P70S6k和p-thr389-P70S6k蛋白在细胞核表达。Western blotting法结果显示二甲双胍作用24 h前、后卵巢颗粒细胞P70S6k、p-thr389-P70S6k、IRS-1及p-ser307-IRS-1蛋白表达有统计学差异(P<0.05),灰度值分析显示添加二甲双胍组P70S6k、p-thr389-P70S6k蛋白表达显著升高,IRS-1及p-ser307-IRS-1蛋白表达显著下降(P<0.05)。结论:二甲双胍可抑制人颗粒细胞P70S6k的表达,并通过Akt/P70S6k/IRS途径调节IRS-1的表达,从而增加颗粒细胞胰岛素的敏感性。
AIM: To investigate the effects of metformin on the phosphorylation of S70S6k and IRS-1 proteins and its ser307 phosphorylation in 40S small subunit of ribosome. Methods: The luteinized granulosa cells of patients with polycystic ovary syndrome (PCOS) were continuously treated with 0.1 mmol / L metformin in vitro for 24 h. The cultured cells without metformin were used as the control group. The expression of P70S6k, IRS-1 and p-ser307-IRS-1 protein were detected by RT-PCR and Real-time PCR, and the expressions of P70S6k, p-thr389-P70S6k, . Results: Real-time PCR results showed that the levels of P70S6k and IRS-1m RNA in the experimental group were significantly lower than those in the control group (P <0.05). The expression of IRS-1 and p-ser307-IRS-1 proteins was detected in the cytoplasm of granulosa cells by immunofluorescence staining, and the expression of P70S6k and p-thr389-P70S6k proteins in the nucleus. The results of Western blotting showed that the expression of P70S6k, p-thr389-P70S6k, IRS-1 and p-ser307-IRS-1 in ovarian granulosa cells were significantly increased after metformin treatment for 24 h (P < The results showed that the expression of P70S6k and p-thr389-P70S6k in metformin group was significantly increased, while the expression of IRS-1 and p-ser307-IRS-1 protein was significantly decreased (P <0.05). CONCLUSION: Metformin inhibits the expression of P70S6k in human granulosa cells and regulates the expression of IRS-1 through Akt / P70S6k / IRS pathway to increase the insulin sensitivity of granulosa cells.