孕酮和雌二醇影响弓形虫入侵和增殖的机制

The mechanism of Toxoplasma gondii-induced reproductive disorders of animal and human has not been elucidated. We hypothesized that dramatic changes of estrogen levels during pregnancy may be a primary cause of high infection rate and pathogenicity of T. gondii during pregnancy. Our previous study showed progesterone and estradiol, which significantly rise during pregnancy, could affect the invasion and proliferation of T. gondii dramatically. Taking this as the starting point, we propose to investigate the mechanisms of progesterone and estradiol directly affect the invasion and proliferation of T. gondii. We will identify the receptors in T. gondii to the two hormones, clarity the role of hormones directly on the effect mode of T. gondii and illustrate effects of the hormones on T. gondii invasion and proliferation, key factors and signal transduction pathways. We will also explore the two hormone-induced target effector molecules of T. gondii, screen host environment regulatory factors of T. gondii in the presence of the hormones, and monitor the effects of these factors on T. gondii reproduction, host immunity, host cellular metabolism, placenta and embryos, to clarify the mechanism of regulatory factors on the regulation of the host environment and then regulate the invasion and proliferation of T. gondii. We expect to reveal the mechanisms of hormones on T. gondii invasion and proliferation, and lay a foundation for further studying the pathogenic mechanisms of T. gondii in pregnancy. This research will provide new ideas for preventing and controlling T. gondii from pregnancy and animal reproductive disorders.

龚地弓形虫诱发动物和人的生殖障碍的机制尚未阐明，我们推测妊娠期机体雌激素水平的巨大变化是影响弓形虫诱发妊娠动物和人高感染性和致病性的重要原因。本课题组前期研究发现孕酮和雌二醇能够显著影响弓形虫的入侵和增殖，本项目以此为切入点，研究孕酮和雌二醇直接影响弓形虫入侵和增殖的作用机制。通过鉴定两种激素的弓形虫受体，明确激素直接作用于弓形虫的效应方式，阐明两种激素影响弓形虫入侵和增殖的途径、关键因子及信号传导通路等；探查两种激素所诱导的弓形虫靶效应分子，从中筛选出弓形虫调节宿主内环境的调控因子，研究调控因子对虫体繁殖、宿主免疫、宿主细胞代谢的影响以及与胎盘和胚胎损伤之间的关系，阐明调控因子对宿主内环境的调节进而调控弓形虫入侵和增殖的机制。研究结果将揭示两种激素直接影响弓形虫入侵和增殖的作用机制，为深入揭示弓形虫对妊娠宿主的致病机制奠定基础，为防控弓形虫引起妊娠动物和人的生殖障碍提供新思路。

龚地弓形虫是重要的人兽共患胞内原虫，对妊娠动物和人具有特殊的致病力和致病作用。我们选择妊娠期间变化较大的雌二醇和孕酮，研究两种激素对弓形虫生命活动的影响和机制。.研究结果显示，雌二醇能够促进弓形虫的入侵和增殖能力，增强弓形虫对小鼠的致病力。雌二醇进入虫体胞质激活cGMP依赖性蛋白激酶G（PKG）和磷酸肌醇磷脂酶C（PI-PLC），引发弓形虫胞内钙离子波动，促进微线蛋白2（MIC2）分泌，提升弓形虫逸出和胞外运动能力，导致弓形虫的增殖能力和对小鼠致病力增强。弓形虫的羟类固醇脱氢酶（HSD）能够催化雌酮生成雌二醇，过表达HSD的虫株对小鼠致病性增强。孕酮能够明显抑制虫体增殖，高水平孕酮致弓形虫对小鼠的致病力下降。孕酮亦能够诱发弓形虫内钙离子释放并促进虫体逸出，但它同时抑制了MIC2分泌，导致虫体逸出后侵入新细胞的能力下降，最终导致弓形虫的致病性降低。在孕酮作用下，可见弓形虫分裂异常、自噬相关蛋白8(ATG8)聚集、胞质出现空泡化、线粒体膜电位下降和虫体凋亡率明显升高等非正常现象。此外，孕酮能够影响TgGST2在虫体内的定位及其在囊泡转运蛋白时的功能，囊泡形态异常。孕酮还影响虫体胆固醇和脂类物质的代谢，孕酮作用下弓形虫的胆固醇摄取量受到明显抑制，虫体内脂滴数量明显增加，蛋白质组学和代谢组学分析显示弓形虫体内溶血磷脂包括溶血磷脂酰胆碱、溶血磷脂酰肌醇和溶血磷脂酰乙胺醇含量增多。在弓形虫中鉴定出的孕酮膜受体（PGRMC），该受体基因缺失后，孕酮对虫体增殖的抑制作用明显降低。明确弓形虫细胞色素P450和PGRMC是互作蛋白，P450具有CYP3A和CYP2B的酶活性，能帮助弓形虫抵抗外源性药物的杀伤作用，过表达P450的虫株对小鼠致病性显著增强；孕酮作用的虫体的P450表达量降低，酶活性降低。由此可见，两种激素作用于虫体的靶点、通路和功能多种多样。.本研究明确孕酮和雌二醇能够不同程度地影响弓形虫入侵、增殖以及对小鼠的致病力，解析了两种激素对弓形虫作用的分子机制。