基于共培养模型研究三氯生对卵巢及胎盘类固醇合成的干扰作用与机制

Triclosan (TCS), a common antimicrobial agent in personal care and household products, has been found widely detectable in human worldwide. Evidence from animal studies and epidemiology surveys indicated that TCS might disrupt reproduction. WHO has included TCS into the list of endocrine disrupting chemicals and speculated that steroidogenesis might be the key point of TCS’s disrupting effect. Based on our established birth cohort in Shandong, we found that prenatal TCS exposure disrupted reproductive hormones in cord blood, which was mediated by placental steroidogenic enzymes. Steroidogenesis in female takes place in ovary and placenta and both require collaboration of two cells or two organs. Due to lack of in-depth study and limitations in study methods, the targets and mechanisms of TCS on ovarian and placental steroidogenesis remain unclear. Current studies suggested that ovarian-predominant and/or placenta-specific miRNAs might be regulatory factors. Therefore, we plan to establish in vitro models for both ovarian and placental steroidogenesis by adopting the co-culture system, which is capable to mimic the inner environment of human body. Based on the population exposure levels, we will detect the concentrations of both intermediate and terminal steroidogenic enzymes as well as the mRNA and protein expression of related steroidogenic enzymes so as to clarify the impacts and targets of TCS on ovarian and placental steroidogenesis. Meanwhile, we will search for microRNAs related to TCS’s reproductive disrupting effects, as well as their target genes and signal pathways in the downstream by a series of biomolecular tests and bioinformatic analysis so as to find out the mechanism of reproductive disruption of TCS. As a whole, the study will provide a scientific basis for clarification of the impacts of TCS exposure on women’s reproductive health, strategy development for prevention and intervention, and ultimately the improvement of women’s reproductive health.

三氯生是个人护理品及日用化学品中常用抗菌剂，已在国内外人群中普遍检出，且研究提示可干扰生殖。WHO已将其列为环境内分泌干扰物，并推测类固醇合成是干扰关键。项目组在出生队列中发现，母体内三氯生经胎盘类固醇合成酶介导后影响脐血性激素。女性类固醇合成发生于卵巢和胎盘，具有两细胞/两器官相互配合的特点。因缺少深入研究，方法局限，目前三氯生对女性类固醇合成的干扰靶点未知,机制不明。有证据提示卵巢/胎盘特异miRNA参与调控三氯生的干扰作用。本项目拟采用模拟人体内环境的共培养系统，建立卵巢和胎盘类固醇合成的体外模型，在人群暴露水平下，检测中间和终末产物浓度及合成酶表达，明确干扰作用，寻找干扰靶点。同时，综合多种分子生物学和生物信息学方法，筛选三氯生生殖干扰相关miRNA，明确下游靶基因及通路，阐明干扰机制。最终为明确日常TCS暴露对女性生殖健康的危害、开展相关预防和干预、提升女性生殖健康提供科学依据。

研究提示，个人护理品及日用化学品中常用抗菌剂三氯生可干扰生殖。据WHO报告提示，类固醇合成是三氯生干扰生殖的关键。项目组在人群研究已发现母体胎盘内类固醇合成受到三氯生干扰。基于此，项目组针对女性类固醇合成的两个靶器官——卵巢和胎盘，进行体外模拟实验，探索三氯生对两细胞配合下的类固醇合成过程的干扰模式和可能机制。首先，项目组参照国外研究构建体外胎盘类固醇合成共培养模型，并自主探索构建体外卵巢类固醇合成共培养模型；基于两个共培养模型，对照两细胞学说开展全面系统的三氯生干扰类固醇合成效应评价；基于此，进一步探索三氯生干扰类固醇合成的调控机制。项目发现，三氯生对胎盘和卵巢两种类固醇合成过程都存在一定程度的干扰，表现为抑制3β-HSD、17β-HSD、P450arom等关键合成酶，进而干扰睾酮、雌二醇等类固醇激素。由此提示，类固醇合成酶可能是三氯生的干扰目标。该发现与项目组前期的人群发现和研究假设相吻合。同时，现有发现提示三氯生对类固醇合成存在多点干扰，进一步的研究须从各点干扰程度与机制进行挖掘。此外，本项目引入共培养模型进行三氯生内分泌干扰效应探索，旨在为探索和解决当前环境内分泌干扰效应与机制研究中的瓶颈提供思路。本研究一方面引入国外研究的胎盘类固醇共培养模型，另一方面同理构建卵巢原代细胞共培养模型。现有结果提示，两个模型能较好反映三氯生的干扰作用，提示其在环境内分泌干扰物的研究中可能有较好的应用前景和价值。