Proteomic Analysis of Placental Extracellular Vesicles Isolated from Maternal Blood by High Resolution Flow Cytometry

通过高分辨率流式细胞术对母血中分离的胎盘细胞外囊泡进行蛋白质组学分析

• 批准号: 9353841
• 负责人: Terry Knud Morgan
• 金额: $ 19.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-19 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353841
• 关键词: Address; Age; Apoptosis; Biogenesis; Biological Markers; Blood; Blood Platelets; Blood Volume; Blood flow; Body mass index; Cell membrane; Cells; Centrifugation; Clinical; Collection; DNA; Data; Encapsulated; Flow Cytometry; Functional Magnetic Resonance Imaging; Functional disorder; Funding; Future; Genomics; Gestational Age; Imaging technology; Linear Regressions; Lipids; Magnetic Resonance Imaging; Measurement; Measures; Methods; Modeling; Monitor; Outcome; Perfusion; Placenta; Placental Growth Factor; Plasma; Pregnancy; Pregnancy Outcome; Prospective cohort; Proteins; Proteomics; Protocols documentation; RNA; Race; Research; Resolution; Sampling; Signal Transduction; Sorting - Cell Movement; Source; Standardization; Stream; Technology; Testing; Time; Ultracentrifugation; United States National Institutes of Health; Vascular Endothelial Growth Factors; Vesicle; Villous; Woman; adverse outcome; adverse pregnancy outcome; base; density; exosome; extracellular vesicles; in vivo; interest; liquid biopsy; novel; novel marker; novel strategies; parity; particle; predictive marker; receptor; submicron; transcriptomics

The ability to monitor the placenta in vivo from early gestation to term is limited and there is immense interest in developing new methods to perform “liquid biopsies” of maternal blood to test for placental dysfunction. Biomarker studies using plasma have been largely unsuccessful to predict adverse outcomes before mid-gestation; and, it is clear that a more sensitive approach to enrich placental signals is needed if we are to understand the early pathophysiology of placental dysfunction and better predict serious complications. Fragments of the placenta are released into the maternal blood stream as early as 6 weeks' gestation. The concentration of these extracellular vesicles (EVs) appears to be related to placental size and pregnancy outcomes. The term EV includes a range of submicron particles, including small exosomes derived from endosomal biogenesis, larger vesicles budding directly from the cell membrane, and fragments of cells undergoing apoptosis. These lipid encapsulated particles contain protein, RNA, and DNA, which are suitable for –omics. There is considerable interest in the potential utility of EVs for biomarker studies, because they provide an in vivo window into placental function. Progress in the field has been limited, however, by the lack of placental-specific EV isolation methods. To address this need, our group will employ a new high resolution flow cytometry (HRFC) sorting method that can reliably identify, quantitate, and purify cell-specific submicron- sized EVs. HRFC provides 10-fold better submicron resolution than currently available flow sorting technology, enabling cell-specific EV isolation and purification down to the exosome range (100nm). We hypothesize that isolating placental EVs from early pregnancy plasma will enrich biomarker signals and provide a new means to reveal novel markers that are otherwise undetectable by conventional proteomic analysis of maternal plasma. We will first validate the HRFC sorting method using placental explant-derived EVs diluted in non-pregnant plasma. We will then test whether enriching placental signals from early (12-14 weeks') and late mid-gestation (26-28 weeks') correlate with clinical outcomes. Our highly novel approach will combine this new placental-specific EV isolation method with state-of-the-art magnetic resonance imaging (MRI) technology funded by the NIH to measure placental size and uteroplacental blood flow in a prospective cohort (U01-HD087182-01). Plasma, total EVs (differential centrifugation), and purified placental EVs isolated from early and late mid-gestation will be banked for proteomic, transcriptomic, and genomic studies. Biomarker concentrations will be compared in plasma, total EVs, and placental-specific EVs. Linear regression modeling will analyze potential relationships between biomarker levels, placental size, and perfusion measurements made by MRI after adjusting for covariates (e.g. age, parity, race, body mass index, and gestational age at the time of blood collection). Proteomic analysis of placental EVs collected at 12-14 and 26- 28 weeks' will compare 12 women who have severe placental dysfunction with 12 closely matched controls.

从妊娠早期到足月，在体内监测胎盘的能力是有限的，而且有巨大的 对开发用于检测胎盘的母血“液体活组织检查”新方法的兴趣 功能障碍。使用血浆进行的生物标记物研究在很大程度上不能成功地预测不良后果。 在怀孕中期之前；很明显，如果我们需要更灵敏的方法来丰富胎盘信号 是为了了解胎盘功能障碍的早期病理生理学，更好地预测严重的并发症。 早在怀孕6周时，胎盘碎片就被释放到母亲的血液中。 这些细胞外小泡(EV)的浓度似乎与胎盘大小和妊娠有关。 结果。术语EV包括一系列亚微米颗粒，包括源自 内体生物发生，直接从细胞膜萌发的较大小泡，以及细胞的碎片 正在经历细胞凋亡。这些脂质包裹的颗粒含有蛋白质、RNA和DNA，这些都是合适的 为了--经济学。电动汽车在生物标记物研究中的潜在效用引起了相当大的兴趣，因为它们 提供了解胎盘功能的体内窗口。然而，该领域的进展受到缺乏的限制 胎盘特异性EV分离方法。为了满足这种需求，我们小组将采用一种新的高分辨率 流式细胞术(HRFC)分选方法可以可靠地鉴定、定量和纯化细胞特异性亚微米- 大小的电动汽车。HRFC提供比当前可用的流动分选技术高10倍的亚微米分辨率， 实现细胞特异性EV的分离和提纯，精确度可达外切体范围(100 Nm)。 我们推测，从妊娠早期血浆中分离胎盘EV将丰富生物标志物信号 并提供了一种新的方法来揭示传统蛋白质组无法检测到的新标记 母体血浆分析。我们将首先使用来自胎盘的外植体来验证HRFC分选方法 EVS在非妊娠血浆中稀释。然后我们将测试是否从早期(12-14)丰富胎盘信号 妊娠中期(26-28周)和晚期(26-28周)与临床结局相关。我们非常新颖的方法将 将这种新的胎盘特异性EV隔离方法与最先进的磁共振成像技术相结合 (MRI)由美国国立卫生研究院资助的技术，用于测量胎盘大小和子宫胎盘血流量 队列(U01-HD087182-01)。分离的血浆、总EVS和纯化的胎盘EVS 从妊娠早期和中期开始，将储存蛋白质组、转录组和基因组学研究。 生物标志物浓度将在血浆、总电动汽车和特定于胎盘的电动汽车中进行比较。线性回归 建模将分析生物标志物水平、胎盘大小和血流灌注之间的潜在关系 在调整协变量(例如，年龄、产次、种族、体重指数和 采血时的胎龄)。12-14和26-14采集的胎盘EVS的蛋白质组学分析 28周后，将有12名严重胎盘功能障碍的女性与12名相近的对照组进行比较。