摘要
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 detect the effect and mechanism of Bisphenol A（BPA）on the expression of P450 arom and estradiol（E2）in human granulosa cells.Methods：A cell culture model was established in vitro.The human granulosa cells were confirmed by immunohistochemical staining.CCK-8proliferation assay was used to analyze the viability of human granulosa cells in vitro.A concentration gradient of BPA（0,0.1,1,10,100mg/L）or BPA＋PPAR-γinhibitor GW9662（1μmol/L）was assigned to process human granulosa cells.ELISA was employed to detect the E2 expression in the culture medium of each group.Then the cell deposits were obtained.Real-time PCR was used to determine the P450 arom mRNA expression level of each group.Results：After3to 5days＇ culture in vitro,the proliferation of human granulosa cells reached the peak.FSHR（Follicle-stimulating hormone receptor）immunocytochemistry showed that the positive staining was located in the membrane and cytoplasm of the cells.The purity of granulosa cells reached95%.The results of ELISA and RT-PCR showed that the P450 arom mRNA level and the E2 expression increased（P〈0.05）when treated with BPA（0.1mg/L）,as compared with the blank group（treated with BPA,0.1μg/ml）.Compared with the blank group,the expression of P450 arom mRNA and E2 decreased after treated with a concentrations gradient of BPA（1,10,100mg/L）（P〈0.05）.When PPAR-γinhibitor GW9662 was added,the expressions of P450 arom mRNA and E2increased（P〈0.05）than in control groups without GW9226 added.Conclusion：BPA may interfere in estradiol production,and GW9662 can reduce the interference of BPA in P450 arom and estradiol prod
出处
《武汉大学学报（医学版）》
CAS
2016年第5期725-729,共5页
Medical Journal of Wuhan University
基金
国家自然科学基金面上项目(编号:81370707)
