Microvesicle Isolation from Mycobacterium tuberculosis infected macrophage using

描述(由申请人提供)：结核病是世界上最流行和最致命的传染病，其传统诊断在艾滋病毒-结核病合并感染者中的特异性较低。最近，细胞外的微囊，包括胞外体，在细胞内的隔室之间运输分子货物已经成为病原体相关诊断的一个有吸引力的标志。微泡不仅携带结核分枝杆菌(Mtb)分泌的抗原，还携带宿主相关标志。尽管微囊分析在开发新的诊断和治疗应用方面具有巨大的潜力，但缺乏有效和标准的分离技术仍然是一个主要的挑战。我们建议采用我们的尖端微流控过滤技术，专门分析受结核杆菌感染的宿主细胞分泌的微囊内的生物成分。微流控体系结构结合了嵌入式堰式过滤屏障，该屏障平行于 流向。在微通道的弯曲路径中产生的横向离心流，产生了一种驱动力，将较小尺寸的部件输送过屏障。这种微流控设计可以最大限度地减少传统过滤分析中经常出现的压降和堵塞问题；因此，系统可以在高流量下连续流动，从而可以轻松地处理大容量样品，如尿液。我们建议设计一种多级过滤系统，它由一系列具有不同屏障间隙的微流控通道组成，以同时分离不同阶段的不同大小的微囊。含有合成脂泡的模拟生物流体将被用于评估我们的微流控系统的效率。我们将在理论计算和实验观测的基础上，对微流控通道进行优化设计。此外，原型多级微流控过滤系统将被用于回收和分析来自健康和结核分枝杆菌感染的巨噬细胞的微囊。由于微泡的产生受到结核分枝杆菌感染的强烈影响，我们将分析收获的微泡的物理性质(大小分布、形态和表面电荷)和蛋白质组学，以区分健康和感染细胞。还将测量常见的外体标记(CD63、CD9、Alix和TSG101)和微泡携带的Mtb抗原(LAM和CFP-10)的浓度。这些初步结果将使我们能够研究分枝杆菌成分在细胞内运输的潜在机制。总之，这项由研究人员发起的小额研究资助可以帮助我们建立微泡分析的方法学，并探索微泡在宿主对结核分枝杆菌感染反应中的潜在作用。这是初步的 研究将使我们了解结核分枝杆菌感染的机制，并进一步开发新的诊断和治疗工具。

DESCRIPTION (provided by applicant): Conventional diagnoses for tuberculosis (TB), the most prevalent and deadliest infectious diseases worldwide, suffer from low specificity in HIV-TB co-infected individuals. Recently extracellular microvesicles, including exosomes, that transport molecular cargo between intracellular compartments have become an attractive marker for pathogen-associated diagnosis. Microvesicles carry not only Mycobacterium tuberculosis (Mtb) secreted antigens but also host associated markers. Although the analysis of microvesicles has great potential to allow development of new diagnostic and therapeutic applications, lack of an efficient and standard separation technique remains a major challenge. We propose to adapt our cutting-edge microfluidic filtration technology to specifically profile biocomponents inside microvesicles secreted by Mtb infected host cells. The microfluidic architecture incorporates an embedded weir-like filtration barrier that is oriented parallel to the flow direction. The transverse centrifugal flow generated within the microchannel's curved path, creates a driving force that transports smaller-sized components across the barrier. This microfluidic design can minimize the problems of pressure drop and clogging usually appearing in conventional filtration assays; hence, the system can be operated with continuous flow at high flow rate so that large volume samples, such as urine, can be easily processed. We propose to design a multi-stage filtration system that consists of a series of microfluidic channels with various barrier gaps to simultaneously isolate different sizes of microvesicles at different stages Simulated biofluids containing synthetic lipid vesicles will be used to evaluate the efficiency of our microfluidic system. We will optimize the design of microfluidic channel based on our theoretical calculations and experimental observations. In addition, the prototype multi-stage microfluidic filtration system will be utilized to recover and analyze microvesicles derived from healthy and Mtb infected macrophages. Because the production of microvesicles is strongly influenced by Mtb infection, we will analyze the physical properties (size distribution, morphology, and surface charges) and proteomics of harvested microvesicles to discriminate between healthy and infected cells. The concentrations of common exosome markers (CD63, CD9, Alix, and TSG101) and Mtb antigens carried by microvesicles (LAM and CFP-10) will also be measured. These preliminary results will allow us to investigate the potential mechanisms of intracellular transport of mycobacterial components. In summary, this Investigator-Initiated Small Research Grant can help us to establish the methodology for microvesicle analysis and to explore the potential role of microvesicles in the host response to Mtb infection. This preliminary study will allow us to understand the mechanism of Mtb infection and further develop new diagnostic and therapeutic tools.