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目的探讨雌激素(E2)对雌激素受体-β(ER-β)与转录因子叉头蛋白3(FoxO3)的表达及其对卵巢颗粒细胞增殖与凋亡的影响。方法利用添加1μmol/L的E2或100 nmol/L ICI182.780的TCM199培养基体外培养卵巢颗粒细胞;采用WST比色法检测不同培养条件下卵巢颗粒细胞的增殖;RT-PCR和Real-time PCR检测ER-β与FoxO3 mRNA的表达,采用细胞免疫荧光和Western blotting检测ER-β与FoxO3的表达。结果 E2能促进卵巢颗粒细胞增殖,而ICI182.780抑制卵巢颗粒细胞增殖;RT-PCR扩增结果与理论值符合,Real-time PCR结果显示,ICI182.780与E2比较,ER-βmRNA表达下调,FoxO3 mRNA表达上调(P<0.05);Western blotting法结果显示,ER-β和FoxO3蛋白表达差异有统计学意义(P<0.05),灰度值分析显示添加E2中ER-β蛋白表达升高,FoxO3蛋白下降(P<0.05),添加ICI182.780中的ER-β表达下调,FoxO3表达上调,与E2比较差异有统计学意义(P<0.05)。结论 E2可提高ER-β的表达,降低FoxO3的表达,影响卵巢颗粒细胞的增殖与凋亡。
Objective To investigate the effect of estrogen on the expression of estrogen receptor-β (ER-β) and transcription factor Fork protein 3 (FoxO3) and on the proliferation and apoptosis of ovarian granulosa cells. Methods Ovarian granulosa cells were cultured in vitro using TCM199 medium supplemented with 1μmol / L E2 or 100nmol / L ICI182.780. The proliferation of ovarian granulosa cells was detected by WST colorimetric assay. RT-PCR and Real-time PCR The expression of ER-β and FoxO3 mRNA was detected. The expression of ER-β and FoxO3 was detected by immunofluorescence and Western blotting. Results E2 could promote the proliferation of ovarian granulosa cells, and ICI182.780 inhibited the proliferation of ovarian granulosa cells. The results of RT-PCR coincided with the theoretical values. Real-time PCR results showed that the expression of ER-βmRNA was down-regulated in ICI 182.780 and E2, The expression of FoxO3 mRNA was upregulated (P <0.05). The results of Western blotting showed that the expression of ER-βand FoxO3 protein had statistical significance (P <0.05). The gray value analysis showed that the expression of ER- FoxO3 protein decreased (P <0.05). The expression of FoxO3 in ICI182.780 was down-regulated and the expression of FoxO3 was up-regulated. The difference was statistically significant compared with E2 (P <0.05). Conclusion E2 can increase the expression of ER-β, reduce the expression of FoxO3 and affect the proliferation and apoptosis of ovarian granulosa cells.