Role of extracellular vesicles in trophoblast invasive migration

胎盘是胎儿正常发育的重要器官，其成功植入取决于滋养细胞对子宫的入侵，直接影响动物和人类的健康，导致重大的福祉和健康老龄化问题。流行病学研究表明，只有约30%的妊娠得到临床认可，而大多数妊娠是自发或因妊娠并发症而丧失的，而着床过程是这一过程的关键调节因素。尽管胎盘对胎儿发育很重要，但我们对介导胎盘着床的机制知之甚少——胎盘着床是发育健康、功能胎盘的关键步骤，但滋养细胞侵入性迁移到接受子宫内膜是至关重要的。这项工作计划将为植入过程中滋养细胞侵袭性运动的过程以及它是如何由周围子宫内膜释放的微泡调节的提供世界领先的新分子机制。外渗滋养细胞（evt）从胎盘锚定绒毛中的细胞滋养细胞增殖和迁移，侵入母体蜕膜和子宫肌层。对细胞运动的重要分子途径知之甚少，但子宫内膜的细胞外囊泡分泌已被证明对这种调节很重要。本项目将剖析子宫内膜通过释放细胞外囊泡来调节滋养细胞侵袭性迁移的分子机制，使用一系列细胞生物学分析方法，这些方法在我们的实验室中经常用于测量细胞运动和侵袭，并确定参与这些过程的因素。细胞外囊泡和局部因子的分离也是常规的。常规使用的人类细胞系最初将从子宫内膜细胞系中分离出EV及其对滋养细胞运动和侵袭的影响，但离体样本也将进行测试。在项目的第一部分，将建立最佳条件，以确定反应最灵敏的滋养细胞和最活跃的子宫内膜细胞外囊泡。研究的第二部分旨在通过使用我们目前在bbsrc资助的工作中进行的协议和方法进行无偏质谱分析，获得微囊泡的完整特征。该项目的最后一部分将旨在描述一些新发现的标记物，并确定它们在细胞外囊泡中的存在是否对通过基因调控或通过使用可用抑制剂（例如抗体和/或化学抑制剂）使用功能获得和功能丧失研究来调节滋养细胞侵袭性迁移很重要。

The placenta is an essential organ for appropriate foetal development and its successful implantation depends upon invasion of the uterus by trophoblast cells, impacting directly on both animal and human health leading to significant well-being and healthy ageing issues. Epidemiological studies have shown that only around 30% of all conceptions get clinically recognized whilst the majority are lost either spontaneously or during pregnancy complication and that the process of implantation is a key regulator in this process. Despite the importance of the placenta to foetal development, we know little of the mechanisms that mediate placental implantation - the key step in developing a healthy, functional placenta, but the invasive migration of the trophoblast into the receptive endometrium is paramount. This proposed programme of work will provide world-leading, novel molecular mechanistic insight to the process of trophoblast invasive motility during implantation and how it is regulated by microvesicles released from the surrounding endometrium.Extravillous trophoblast cells (EVTs) proliferate and migrate from the cytotrophoblast in the anchoring villi of the placenta and invade the maternal decidua and myometrium. Little is known about the molecular pathways that are important for cellular motility but extracellular vesicle secretion by the endometrium has been shown to be important for such regulation.This project will dissect the molecular mechanisms by which the endometrium regulates trophoblast invasive migration through the release of extracellular vesicles using an array of cellular biology assays that are routinely used in our laboratories to measure cell motility and invasion, and determine the factors involved in these process. The isolation of extracellular vesicles and the factors that are localised within is also routinely done. Human cell lines routinely used will initially be used from endometrial linage to isolate EV and their effects on trophoblast cells for motility and invasion but ex vivo samples will also be tested.In the first part of the project, the best conditions will be established to determine the most responsive trophoblast cells and the most active endometrial extracellular vesicles. The second part of the study will aim to get a full signature of the microvesicles through an unbiased mass spectrometry analysis using protocols and approaches that have been conducted within our current BBSRC-funded work. The last part of the project will aim to characterise some of the newly identified markers and determine whether their presence in extracellular vesicles are important to regulate the trophoblast invasive migration through the use of gain of function and loss of function studies either by gene regulation or through the use of available inhibitors (e.g. antibodies and or chemical inhibitors).