The Effect Of Metformin On Polycystic Ovarian Syndrome Luteinizing Granulosa Cells In IRS-1 And Phosphorylation Expression

Objective:1.Isolated and cultured in vitro with polycystic ovary syndrome(polycystic ovarian syndrome PCOS) and non-PCOS patients luteinizing granulosa cells with follicle stimulating hormone receptor(Follicle Stimulating Hormone, FSHR) immunofluorescence;2.RT-PCR and real-time PCR, Immunofluorescence and Western-blot chemical methods for detecting Metformin(Metformin, Met) on luteinizing granulosa cells of the insulin receptor substrate-1(insulin receptor substrate IRS-1)and p-ser307-IRS-1 messenger RNA(Messenger RNA m RNA) and protein expression.MethodsSeparation and purification vitro PCOS and non-PCOS group luteinizing granulosa cells were identified by staining FSHR immune cells; set Add Met treated cultures as the experimental group,insulin-like growth factor adding-1(insulin-like growth factor I, IGF-1) is positive control group, 15% FBS-M199 medium supplemented with Met with PBS as a negative control group training; RT-PCR and by expressing real-time PCR detection and analysis of IRS-1m RNA cell immunofluorescence method for qualitative and Western-Blot analysis of quantitative detection of IRS-1 and p-ser307-IRS-1 protein expression.ResultsFSHR protein immunocytochemistry is identified granulosa cell specific marker, FSHR positive staining localized in the cytoplasm and cell membrane,green fluorescent nuclei blue fluorescence microscope FSHR positive rate greater than 95%,indicating that the separation cultured granulosa cell purity of more than 95%.By RT-PCR amplified fragment size IRS-1 is 134 bp, real-time PCR analysis showed a significant reduction Add Met granulosa cells IRS-1 m RNA expression Luteinized PCOS group,and the positive andnegative control group,the difference was statistically significance(p <0.05); IRS-1 p-ser307-IRS-1 protein expression in cells by immunofluorescence detection and localization in granulosa cell cytoplasm; Western-blot detection Met after 24 h ovarian luteinizing granulosa cells IRS-1 and p- ser307-IRS-1 protein downregulation, and the difference was statistically significant(p <0.05), the gray value analysis showed that women with PCOS ovarian luteinizing granulosa cells IRS-1 and p-ser307-IRS-1 protein than non-PCOS group; add the Met group action 24 h PCOS group luteinizing granulosa cells IRS-1 and expression of p-ser307-IRS-1 protein was significantly decreased(p <0.05), and non-PCOS patients was significantly different.Conclusion1.Isolation FSHR immunofluorescence staining granulosa cell protein ratio greater than 95%, more than 95% purity identification2.PCOS patients with ovarian granulosa cells phosphorylation of IRS 1-expression and increased3.Met reduce PCOS ovarian granulosa cells phosphorylation of IRS-1 and its expression...