二噁英通过干扰线粒体相关蛋白影响人胎盘类固醇激素生成的机制初探

Reproductive and endocrinology damages are serious and sensitive effects of dioxins. The steroidogenesis function of placenta plays an important role in the maintenance of pregnancy. In the preliminary study, the interference of the steroidogenesis has been proved in the human placenta, but the mechanisms are unclear. We hypothesized that dioxins may affect mitochondrial proteins associated with cholesterol transportation in the placenta and thus interfere with steroid generation. The human placentas of subjects and trophoblast cell lines (Bewo) will be applied in the present study. Concentrations of dioxins in placentas were measured using ID/HRGC/HRMS. The generation of progesterone and pregnenolone was analyzed in the placental mitochondrial; The expression and interaction of proteins in the placental mitochondrial were compared and explored in subjects with different levels of dioxins, including the transduceosome complexes (TSPO, VDAC, etc.) and fusion / fission-related proteins (MFN1/2, OPA1, etc.). Dynamic changes of detection mitochondrial shaping will be detected in Bewo cells using Drift Microscope System; The expression of target proteins were evaluated by the siRNA targeting TSPO / MFN2 mRNA and the treatment of 22(R)- hydroxycholesterol. Overall, we focus on the mechanisms of TCDD affecting the transduceosome and fusion-related proteins, providing new clues and basis for mechanisms on steroidogenesis interference after the exposure to dioxins.

生殖内分泌损害是二噁英严重和敏感的效应终点之一，胎盘作为主要的内分泌器官，其分泌类固醇激素对于维持妊娠具有重要作用。本项目组前期研究证明二噁英干扰人胎盘类固醇激素合成，但机制尚不清楚。本研究假设其可能影响胎盘线粒体中与胆固醇转运相关的蛋白，拟应用典型区域的内暴露机制研究与人永生化Bewo滋养细胞株体外研究对假设进行验证。高暴露和低暴露孕妇分娩后取胎盘，测定二噁英类物质浓度；提取胎盘线粒体，分析孕酮和孕烯醇酮的生成；比较线粒体中“转导相关复合物”(TSPO、VDAC等)和“融合与裂变相关蛋白”(MFN1/2、OPA1等)的表达和相互作用。在Bewo细胞中，染毒后检测活细胞线粒体融合与分裂动态改变；运用siRNA干扰和22(R)-羟基胆固醇处理，比较激素生成，目标蛋白及CYP11A1、3betaHSD等关键酶表达的差异。由此验证假设，为二噁英的胎盘毒性和生殖内分泌损害提供理论依据。

作为典型的内分泌干扰物和持久性有机污染物，二噁英类物质广泛存在于环境和人体中。生殖和内分泌毒性是二噁英类物质最敏感的效应终点。本研究应用典型区域的体内暴露机制研究与人永生化Bewo滋养细胞株体外研究对假设进行验证。在Bewo细胞中，染毒后检测活细胞线粒体融合与分裂动态改变；比较激素生成，目标蛋白及CYP11A1、3betaHSD等关键酶表达的差异。高暴露和低暴露孕妇分娩后取胎盘，测定二噁英类物质浓度；提取胎盘线粒体，分析孕酮和孕烯醇酮的生成；比较线粒体中“转导相关复合物”(TSPO、VDAC等)和“融合与裂变相关蛋白”(MFN1/2、OPA1等)的表达和相互作用。.研究结果显示：.1. TCDD能够对BeWo细胞的增殖产生影响，并具有剂量、时间效应关系，随染毒的浓度增加以及随着染毒的时间增加，细胞增殖逐渐下降。TCDD能够下调BeWo细胞MFN1/2并上调DRP1的表达，破坏线粒体融合分裂功能；同时通过上调TSPO、ACBD3以及下调ATAD3来对BeWo线粒体膜胆固醇转运蛋白复合物产生影响，降低了类固醇激素生成的速率。.2. TCDD能够改变FIS1、MFN1、MFN2、DRP1、OPA1、VDAC2、ATAD3启动子区域特定位点的甲基化水平，通过表观遗传学修饰影响基因功能。.3. 高暴露地区孕妇分娩时胎盘样本中PCDD/Fs暴露水平远远高于居民对照组。PCDD/Fs暴露会对氯化工人全基因组DNA甲基化水平造成影响，随着PCDD/Fs内暴露水平的增加，DNA甲基化水平出现下降。.4. PCDD/Fs暴露水平不同，对胎盘组织中线粒体相关蛋白造成影响，其中FIS1、MFN1、DRP1、OPA1蛋白的表达水平，均随着PCDD/Fs暴露水平的升高而升高。.本项目的研究结果为二噁英的胎盘毒性和生殖内分泌损害提供了理论依据，为后续人群健康研究提供数据基础。