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目的:研究双酚A(BPA)对人卵巢颗粒细胞细胞色素P450芳香化酶(P450arom)和雌二醇(E2)表达的影响机制。方法:提取人卵巢颗粒细胞进行体外培养,细胞鉴定采用免疫细胞化学法,CCK-8增殖实验分析细胞体外增殖活力。予以不同浓度BPA(0,0.1,1,10,100mg/L)或BPA+PPAR-γ抑制剂GW9662 1μmol/L处理处理人卵巢颗粒细胞,ELISA法测定各组E2浓度,获取细胞沉淀,RT-PCR法测定P450arom mRNA表达。结果:人卵巢颗粒细胞体外培养第3-5天达增殖分裂高峰,FSHR免疫细胞化学鉴定显示阳性染色定位于细胞膜和细胞质,细胞纯度达95%以上,ELISA及RT-PCR结果显示当BPA浓度为0.1mg/L时,P450arom mRNA及E2生成较空白对照组增加(P<0.05),当BPA浓度为1,10,100mg/L时,P450arom mRNA及E2生成逐渐减少(P<0.05),加入GW9662后,P450arom mRNA及E2表达量较单独BPA处理组增高(P<0.05)。结论:BPA干扰人卵巢颗粒细胞E2的生成,该干扰可能通过PPAR-γ核受体途径进行。
Objective: To study the mechanism of BPA on the expression of cytochrome P450 aromatase (P450arom) and estradiol (E2) in human ovarian granulosa cells. Methods: Human ovarian granulosa cells were isolated and cultured in vitro. Immunocytochemistry and CCK-8 proliferation assay were used to analyze the proliferation of human ovarian granulosa cells in vitro. Human ovarian granulosa cells were treated with different concentrations of BPA (0, 0.1, 1, 10, 100 mg / L) or BPA + PPAR-γ inhibitor GW9662 1 μmol / L, E2 concentrations were measured by ELISA, cell pellet was obtained and RT- Method to determine P450arom mRNA expression. Results: Human ovarian granulosa cells reached the peak of proliferation and proliferation on day 3-5, and the immunofluorescence staining of FSHR showed that the positive staining was located on the cell membrane and cytoplasm. The purity of the cells was over 95%. ELISA and RT-PCR results showed that when BPA concentration was When the concentration of BPA was 1, 10, and 100 mg / L, P450arom mRNA and E2 production gradually decreased (P <0.05). After addition of GW9662 , P450arom mRNA and E2 expression increased compared with BPA alone group (P <0.05). Conclusion: BPA interferes with the production of human ovarian granulosa cells E2, which may be mediated by the PPAR-γ nuclear receptor pathway.