Extracellular vesicles - Characterising the structure-function relationships of extracellular vesicles in cell communication within the neurovascular

细胞外囊泡 - 表征神经血管内细胞通讯中细胞外囊泡的结构-功能关系

• 批准号: 2431354
• 金额: --
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2431354
• 关键词: Extracellular; vesicles; Characterising; structure; function

"The nature of cell communication in a multicellular organism may result in health or disease. Within the central nervous system (CNS), it is not fully understood how the mix of cells (neuronal, non-neuronal and immune cells) communicate, nor the language that is used. The change in this communication in age-associated conditions is also poorly understood.Over recent years, extracellular vesicles (EV) have been identified as a novel intercellular communication mechanism1. These membrane bags, released from cells during health, disease and cell death, carry many factors to recipient cells to elicit responses (desirable and non-desirable). There are paradoxical data to suggest EV may be both neuroprotective and neurotoxic2. This highlights the need for detailed EV analysis from each of the CNS cell types so we may understand EV uptake, function and how they change in age-associated conditions. Preliminary work reveals that EV carry a large range of factors that may underpin their activity, including a family of active enzymes which may help to control inflammation and repair responses2. This project seeks to identify and characterise the key factors associated with EV, that mediate their function. The aims of this project are: to generate cells of the neurovascular unit from induced pluripotent stem cells and assess EV release in mono- and co-culture to assess the ability of EV to induce responses on other cells of the neurovascular unit (e.g. inflammatory responses) to define the molecular mediators of function through detailed MS analysis to define EV surface proteins mediating intercellular communication to investigate EV-associated enzyme activity that controls inflammation. identify key proteins present on the surface of EV that mediate EV function in interacting with macrophages and modulating the innate immune response. This will work from a large dataset of mass-spec results to identify lead molecules for further study. to define the function of prioritised proteins in EV activity. Using a variety of cell biological and molecular approaches, this will assess proteins as key ligands for EV binding, uptake or active function.Techniques that will be utilised during the project: Analysis of extensive proteomics data: to identify lead targets for further analysis using systems biology approaches. Cell culture: Isolation of primary cells from human peripheral blood; Tissue culture of a range of cell lines and primary cells. Induced pluripotent stem cell culture to derive functional neuronal networks, astrocytes and microglia. Vesicle analyses: isolation and analysis of EV structure and function using tunable resistive pulse sensing and flow cytometry. Analysis of immune-modulating capability using chemoattraction assays and qPCR. Imaging and analysis: Flow cytometry, light and fluorescence microscopy and automated cell imaging. Cell assays: Induction and analysis of cell death, binding and phagocytosis assays. Mass spectrometry: for the analysis of EV proteome.References:1. Van Niel, G., D'Angelo, G., & Raposo (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews: Molecular Cell Biology: DOI: 10.1038/nrm.2017.1252. Shi, M., Sheng, L., Stewart, T., Zabetian, C.P. & Zhang, J. (2019). New Windows into the brain: CNS-derived extracellular vesicles. Progress in Neurobiology: DOI: 10.1016/j.pneurobio.2019.01.0053. Devitt, A., Griffiths, H.R. & Milic I. (2018). Communicating with the dead: lipids, lipid mediators and extracellular vesicles. Biochemical Society Transactions: DOI: 10.1042/BST2016477"

“多细胞生物体中细胞通讯的性质可能导致健康或疾病。在中枢神经系统（CNS）中，细胞（神经元，非神经元和免疫细胞）的混合如何进行交流以及使用的语言尚未完全了解。近年来，细胞外囊泡（EV）已被确定为一种新的细胞间通讯机制1。这些膜袋在健康、疾病和细胞死亡期间从细胞释放，将许多因子携带到受体细胞以引起反应（期望的和不期望的）。有矛盾的数据表明EV可能既有神经保护作用又有神经毒性2。这强调了对每种CNS细胞类型进行详细EV分析的必要性，因此我们可以了解EV摄取，功能以及它们在年龄相关疾病中的变化。初步研究表明，EV携带大量可能支持其活性的因素，包括一系列可能有助于控制炎症和修复反应的活性酶。该项目旨在确定和验证与EV相关的关键因素，介导其功能。该项目的目标是：从诱导的多能干细胞产生神经血管单位的细胞，并评估在单一培养物和共培养物中的EV释放，以评估EV诱导神经血管单位的其他细胞的反应的能力（例如炎症反应），以通过详细的MS分析确定功能的分子介导物，以确定介导细胞间通讯的EV表面蛋白，从而研究EV-与控制炎症的酶活性相关。鉴定EV表面上存在的介导EV与巨噬细胞相互作用和调节先天免疫应答的关键蛋白。这将从一个大的质谱结果数据集工作，以确定进一步研究的铅分子。以确定EV活动中优先蛋白质的功能。利用各种细胞生物学和分子生物学方法，这将评估蛋白质作为EV结合，摄取或活性功能的关键配体。项目期间将使用的技术：分析广泛的蛋白质组学数据：确定使用系统生物学方法进行进一步分析的主要目标。细胞培养：从人外周血中分离原代细胞;一系列细胞系和原代细胞的组织培养。诱导多能干细胞培养以衍生功能性神经元网络、星形胶质细胞和小胶质细胞。 囊泡分析：使用可调电阻脉冲传感和流式细胞术分离和分析EV结构和功能。使用化学吸引测定和qPCR分析免疫调节能力。成像和分析：流式细胞术，光学和荧光显微镜和自动细胞成像。细胞测定：细胞死亡的诱导和分析、结合和吞噬测定。质谱法：用于EV蛋白质组分析。参考文献：1。货车尼尔，G.，德安杰洛湾& Raposo（2018）.揭示细胞外囊泡的细胞生物学。Nature Reviews：Molecular Cell Biology：DOI：10.1038/nrm.2017.1252.施，M.，Sheng，L.，斯图尔特，T.，Zabetian，C.P. & Zhang，J.（2019）.大脑的新窗口：CNS衍生的细胞外囊泡。Progress in Neurobiology：DOI：10.1016/j.pneurobio.2019.01.0053. Devitt，A.，格里菲思，H.R. & Milic I.（2018年）。与死者沟通：脂质，脂质介质和细胞外囊泡。Biochemical Society Transactions：DOI：10.1042/BST 2016477”