双酚A下调芳香化酶的表达在诱导多囊卵巢综合症发生中的机制研究

Bisphenol A (BPA) which is ubiquitous in the environment is a high-volume-production monomer used in polycarbonated plastic. It is one of the hotly debated endocrine-disrupting-chemicals(EDCs).BPA can be detected in the majority of individuals. There are a lot of reports concerning the effects of BPA on formation and function of female reproductive organs..Polycystic ovary syndrome (PCOS) is a complex genetic disease that affects approximately 10% of women in reproductive age worldwide. Due to heterogeneity and complexity, PCOS cannot be exhaustively explained by any one of reasons. Recently, a cross-sectional study showed that PCOS patients had significant higher BPA blood level compared to the corresponding control group. And multiple cystic follicles in the ovary were found in the rats exposed to BPA(Fig1)..In our early research, we found BPA may dose-dependently downregulate cytochrome P450 aromatase (P450arom) expression in human granulose cells(GCs) (Fig3). The P450arom is the terminal enzyme responsible for the formation of estrogens from androgens. Downregulation of P450 arom may result in dysfunction of estrogen biosynthesis and andogen accumulation, thus hyperandrogenism is established. This resembles clinical endocrinology and metabolism characteristics of patients with PCOS. And during our small numbers of epidemiological study,we discovered that PCOS patients show higher level of BPA than the controls in follicular fluid(Fig2). This demonstrated BPA might play a potential role in PCOS pathophysiology. So we design our experiment on different levels (epidemiological survey and analysis, cell culture, and animal model) to illustrate the role of BPA in the pathogenesis of PCOS. Interestingly, we discovered that estrogen receptor(ER) inhibitor ,ICI 182 780, cannot block BPA's downregulating effect(Fig5),but androgen receptor(AR) inhibitor, Flutamide, can(Fig6). This may illustrate BPA downregulates P450 aroma via AR. This is different from traditional viewpoint:BPA is an estrogen analogue and acts on ER. So later we take advantages of RNAi technique to demonstrate BPA regulate the expression of P450 arom via ER or AR. And with the use of luciferase reporter assay, we could demonstrate the transcriptional activity of BPA, and how does it interfere with natural hormones in the body. And the applications of bioinformation analysis and chromatin immunoprecipitation ,help us find out some BPA-response-element in the promoter of P450 arom, and by the use of site-directed mutagenesis, we might determine the key point of the BPA-response-element in the promoter of P450. That will clearly explain how BPA directly regulates gene transcription and induces PCOS. This may ,from the environment point, provide a new way of thinking about the pathogenesis of PCOS and how to take precautions against the occurrence of PCOS.

双酚A（BPA）是一种广泛存在于环境的内分泌干扰物。有研究提示BPA能引起鼠卵巢呈多囊样改变；我们的前期研究观察到BPA可下调卵巢颗粒细胞中雄激素代谢限速酶-芳香化酶的表达，导致高雄激素血症。由此推测BPA可能与多囊卵巢综合征（PCOS）发生相关。本项目将进一步明确BPA、芳香化酶、PCOS之间的关系及相互作用机理。利用细胞培养模型和RNA干扰技术，确定BPA通过何受体影响芳香化酶的表达；通过质粒构建转染、荧光素酶报告技术，阐明BPA转录激活相应受体的能力及其影响体内正常激素发挥作用的可能机制；通过生物信息学技术、染色质免疫共沉淀技术验证BPA所作用的转录因子与芳香化酶编码基因启动子区DNA中可能存在的BPA反应原件的结合关系。研究结果揭示BPA引起PCOS高雄激素血症的可能机制，解释部分PCOS发生的可能原因，为从环境角度降低PCOS发生提供新思路，并为治疗提供新靶点。

双酚A（BPA）是一种环境内分泌干扰物，广泛存在于日常生活中，对生殖系统影响严重。我们通过临床病例证实与因管性或男性原因行IVF病人相比，PCOS病人外周血及卵泡液中BPA含量显著升高，且高浓度BPA可引起月经周期延长、基础LH/FSH比值升高、基础TT升高、窦卵泡数增多等临床症状，卵泡液中的BPA水平与A、总TT、游FAI及SHBG呈正相关。在动物模型水平，我们通过皮下注射BPA的方法构建BPA暴露的大鼠模型，发现95% BPA诱导的大鼠呈现持续动情状态，卵巢组织呈PCO样病理改变，血清中雄激素水平升高，LH/FSH比值升高等改变，这表明BPA对个体生殖内分泌具有显著影响。在体外细胞水平，我们发现BPA能影响体外培养颗粒细胞性激素的分泌，显著增加雄激素在培养基中的含量，并发现BPA以浓度和时间依赖性地抑制芳香化酶的表达和活性。我们用雌激素核受体阻断剂和雄激素受体阻断剂分别阻断ERs和ARs，发现后者能阻断BPA引起的芳香化酶表达下调，提示BPA通过ARs抑制芳香化酶的表达和活性引起颗粒细胞雄激素合成和分泌增加。为进一步研究AR如何介导BPA下调CYP19A1基因的下调，我们分析启动子区域可能存在的功能性ARE片段，并设计改功能性片段特异的引物序列，构建表达AR的腺病毒感染原代颗粒细胞模型，用不同浓度的BPA（伴有或不伴有雄激素）刺激细胞，并用特异性抗体AR进行染色质免疫共沉淀（ChIP），获取与雄激素受体AR特异性结合的DNA片段，通过定量ChIP实验发现BPA可以浓度依赖性地抑制雄激素诱导的AR与功能性ARE片段的结合，从而影响颗粒细胞CYP19A1的表达，引起颗粒细胞的高雄激素的分泌。进一步我们利用表达芯片探究不同表型病人的颗粒细胞在PCOS不同表型发展中的作用，得到92具有显著表达差异的DEGs。在功能分布上，他们主要与生殖系统疾病，内分泌系统疾病，代谢系统疾病等相关。DEGs的下游功能分析结果提示高BPA暴露的颗粒细胞呈脂质合成激活状态，其中涉及甾体激素的生成与合成和萜类化合物的合成均处于激活状态，其中甾体激素及脂质代谢在PCOS发生中具有挥重要作用。另外，我们定量分析39例PCOS和30例管性不育患者颗粒细胞基因组DNA整体甲基化水平，发现BPA高含量的PCOS患者的颗粒细胞整体甲基化水平显著低于对照组，提示高浓度BPA暴露可增加基因组的不稳定性。