助力美国梅奥诊所在脑部疾病研究取得重大突破

Author: admin     Date: August 25, 2021

梅奥医学中心（Mayo clinic），于1863年在美国创立。它是以不断创新的医学教育和世界领先的医学研究为基础，建立起来的全美规模最大、设备最先进的综合性医疗体系。

每一种活细胞都会释放细胞外囊泡，其在细胞间信号传导和其它生物功能中发挥非常重要的作用。细胞外囊泡潜在的诊断和治疗应用已经得到广泛认可。然而细胞外囊泡的分子机制研究的尚不够成熟，这里介绍一种利用外部电场程序性控制细胞外囊泡和它们装载货物释放的通用方法。原理认证，用培养的大鼠星形胶质细胞来证明外部电刺激的频率如何选择性影响细胞外囊泡的释放，他们的表面蛋白和microRNA分型。这个方法可以广泛的影响生命科学和医学应用。首先，它提出了一个有趣的问题，外部电场活动如何影响细胞外囊泡的产生；第二，它提供了一种调控治疗性电场刺激可能会对治疗脑部疾病有效的新型机制；第三，它提供了一种通过调控电场刺激参数来获得携载相应货物的细胞外囊泡的新方法。不像那些产生细胞外囊泡的化学方法，电场刺激是一种干净的物理方法，它的参数包括刺激频率，电场强度以及波形形态都是可调节的。

AQP4是中枢神经系统主要的水通道蛋白，位于星形胶质细胞的足突上。不加电场刺激时，细胞外囊泡的粒径分布以及AQP4的分布情况。

To characterize EVs in detail, they purified collected EVs using ultrafiltration combined with size-exclusion chromatography to separate EVs from extrinsic proteins, and then used a nano-flow cytometer (N30 Nanoflow Analyzer, NanoFCM Inc., Xiamen, China) to quantify EVs by their size and surface proteins. In this case, we monitored the astrocyte-specific water channel, aquaporin-4 (AQP4), by using an Alexa Flour 488 conjugated monoclonal AQP4 extracellular domain-specific IgG (Figure 1D). We used AQP4 as an indicator for particle numbers and sizes corresponding to astrocytic EVs collected at different stimulation frequencies. In control condition without applied electrical stimulation, the nano-flow cytometry plot shows populations of AQP4-positive EVs of two distinct sizes (Figure 1E).

纳米流式检测仪可以敏锐的捕捉到不同频率的电场刺激下，细胞外囊泡中的外泌体和微囊泡的变化情况。

In this experiment, they found that the size distributions of AQP4-positive EVs are differentially affected by the frequency of electrical stimulation (Figure 2). Stimulation at 2 Hz produces a near uniform distribution of EVs, with the peak of size distribution at ~70 nm, slightly shifted to the right (larger) by comparison with the smaller sized presumptive exosome population collected without electrical stimulation. The major population of AQP4-positive exosomes arising from 2 Hz stimulation was more AQP4-bright than at baseline, supporting that 2 Hz stimulation generated a population of exosomes with increased membrane AQP4. Stimulation at 20 Hz produced fewer EVs of both large and small size than seen in basal conditions, particularly fewer presumptive exosomes. Electrical stimulation at 200 Hz had the opposite effect of 2Hz stimulation. Instead of yielding exosomes, microvesicles-sized EVs dominated (peak size distribution at ~ 170nm) and this population was also enriched in membrane AQP4.