Serrano-Heras Gemma, Díaz-Maroto Inmaculada, Castro-Robles Beatriz, Carrión Blanca, Perona-Moratalla Ana B, Gracia Julia, Arteaga Sandra, Hernández-Fernández Francisco, García-García Jorge, Ayo-Martín Oscar, Segura Tomás
Research Unit, Complejo Hospitalario Universitario de Albacete, Laurel, s/n, CP: 02008, Albacete, Spain.
Department of Neurology, Complejo Hospitalario Universitario de Albacete, Albacete, Spain.
Biol Proced Online. 2020 Aug 1;22:17. doi: 10.1186/s12575-020-00130-8. eCollection 2020.
Improper regulation of apoptosis has been postulated as one of the main factors that contributes to the etiology and/or progression of several prevalent diseases, including ischemic stroke and neurodegenerative pathologies. Consequently, in the last few years, there has been an ever-growing interest in the in vivo study of apoptosis. The clinical application of the tissue sampling and imaging approaches to analyze apoptosis in neurological diseases is, however, limited. Since apoptotic bodies are membrane vesicles that are released from fragmented apoptotic cells, it follows that the presence of these vesicles in the bloodstream is likely due to the apoptotic death of cells in tissues. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients with ischemic stroke and neurodegenerative diseases.
Since there is no scientific literature establishing the most appropriate method for collecting and enumerating apoptotic bodies from human blood samples. Authors, here, describe a reproducible centrifugation-based method combined with flow cytometry analysis to isolate and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis, Parkinson's disease and also in healthy controls. Electron microscopy, dynamic light scattering and proteomic characterization in combination with flow cytometry studies revealed that our isolation method achieves notable recovery rates of highly-purified intact apoptotic bodies.
This easy, minimally time consuming and effective procedure for isolating and quantifying plasma apoptotic bodies could help physicians to implement the use of such vesicles as a non-invasive tool to monitor apoptosis in patients with cerebrovascular and neurodegenerative diseases for prognostic purposes and for monitoring disease activity.
细胞凋亡调控异常被认为是导致包括缺血性中风和神经退行性病变在内的几种常见疾病的病因和/或病情进展的主要因素之一。因此，在过去几年中，对细胞凋亡的体内研究兴趣与日俱增。然而，用于分析神经疾病中细胞凋亡的组织采样和成像方法的临床应用有限。由于凋亡小体是从破碎的凋亡细胞中释放出来的膜泡，因此血液中这些小泡的存在可能是由于组织中细胞的凋亡死亡所致。因此，我们建议使用循环凋亡小体作为生物标志物，来测量缺血性中风和神经退行性疾病患者的凋亡死亡情况。
由于尚无科学文献确定从人类血液样本中收集和计数凋亡小体的最合适方法。本文作者描述了一种可重复的基于离心的方法，并结合流式细胞术分析，以分离和定量缺血性中风、多发性硬化症、帕金森病患者以及健康对照者的血浆凋亡小体。电子显微镜、动态光散射和蛋白质组学表征与流式细胞术研究相结合表明，我们的分离方法能够实现高纯度完整凋亡小体的显著回收率。
这种分离和定量血浆凋亡小体的简便、耗时短且有效的方法，有助于医生将此类小泡作为一种非侵入性工具加以应用，以监测脑血管和神经退行性疾病患者的细胞凋亡情况，用于预后评估和监测疾病活动。
