Effects Of Berberine On Cho With GSK3? Overexpression In Steroid Hormone

Objective:Chinese hamster ovary cells by detecting glycogen synthase kinase-3?(GSK3?)overexpressing(CHO)phenotypic abnormalities,abnormal activity of GSK3? to investigate the effects of CHO androgen secretion.Then CHO cells overexpressing GSK3? given berberine intervention was observed after the intervention of its hormone changes.By changing the experimental test proved abnormal GSK3? phosphorylation and activity is a risk factor for PCOS,and berberine possibly through the regulation of GSK3? reduce androgen secretion CHO cells for clinical application of berberine treatment of PCOS provide the basis for a theoretical basis.Design and Methods:In this study,RT-PCR method to amplify human GSK3p gene construct GSK3p gene eukaryotic expression vector pEGFP-N1-Gsk3?,the CHO cells were subcultured using liposome technology allows GSK3? gene in CHO cells transiently effective expression.Observe the growth state of CHO cells transfected after GSK3?,using Western blot(Western blot)technology assessment GSK3? expression cells,and the supernatant was detected by cell radioimmunoassay of estradiol(E2),progesterone(P),testosterone(T)levels,berberine intervention GSK3?overexpressing CHO cells,the level of detection of the three hormones in the supernatant again,before and after the Rt-PCR to detect the CHO cells transfected GSK3? gene CYP11,CYP17,mRNA expression levels in STAR's.Results:1.The recombinant plasmid pEGFP-N1-Gsk3?,quantitative UV spectroph-otometer,record plasmid concentrations.Omega's LipofectamineTM 2000 reference manual for transfection.24h after transient expression CHO cells was observed by Western Blot to detect cell GSK3? protein expression level was found in comparison with the blank group,the cells transfected GSK3? expression level of the protein is elevated GSK3?.2.Through the culture medium of each group for statistical analysis hormone levels,pEGFP-N1-Gsk3? comparison with pEGFP-Nl:broth T,P,the value of E2 are reduced,but these changes are not statistically significant,after PMSG stimulation,still with statistical significance;pEGFP-N1-Gsk3?+Ber compared with the pEGFP-N1-Gsk3?,the culture medium did not change the value of T,P and E2 are both increased,but not statistically significance.3.RT-PCR determination of cell androgen biosynthesis genes STAR,CYP11,mRNA levels of CYP17 found with empty plasmid group phase,Gsk3?over-expression of STAR expression group was significantly increased,while the expression after berberine treatment STAR have significantly reduced the amount.However,CYP11 and CYP17 expression levels are not significantly changed between the groups.Conclusions:1.Overexpression caused a rise STAR GSK3? gene mRNA,indicating that GS.K3? could affect the expression of steroid hormone.2.Group compared with GSK3? overexpression after berberine treatment STAR gene mRNA expression levels significantly decreased steroid hormone synthesis medicine berberine on GSK3? overexpressing CHO cells have a regulatory role.3.GSK3? gene may be associated with the pathogenesis of PCOS-related and could be targets of a berberine treatment of PCOS.