Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.
Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.
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基于聚合物的沉淀保留了子宫内膜细胞系细胞外囊泡的生物活性。
DOI：
10.1371/journal.pone.0186534
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Yeung WSB
中科院分区：
文献类型：
--
作者：
Niu Z;Pang RTK;Liu W;Li Q;Cheng R;Yeung WSB
细胞外囊泡 (EV) 是细胞释放的膜结合囊泡，充当蛋白质、小 RNA 和 mRNA 转移到远处位点的介质。它们可以通过不同的方法分离。然而，纯化的EV的生物活性却很少被研究。在本研究中，我们比较了超速离心 (UC)、超滤 (UF)、聚合物沉淀 (PBP) 和 PBP 与基于尺寸的纯化 (PBP+SP) 从人子宫内膜细胞和小鼠子宫腔液 (ULF) 中分离 EV 的情况。电子显微镜显示，在测试方法中分离出的 EV 的直径相似。 UF 回收的 EV 数量最多，其次是 PBP，而 UC 和 PBP+SP 的效率明显较低（P<0.05）。根据EV数量与蛋白质的比率，PBP的蛋白质污染最少，显着优于其他方法（P<0.05）。所有分离的 EV 均表达富含外泌体的蛋白 CD63、TSG101 和 HSP70。将滋养层 JEG-3 细胞与等量的通过研究方法分离的荧光标记的 EV 一起孵育，结果表明许多 PBP-EV 处理的细胞呈荧光阳性，但在 UC 和 UF-EV 处理组中只有少数细胞被标记。此外，与其他 3 种方法相比，PBP-EVs 可以将更多的 miRNA 转移到受体细胞中（P<0.05）。 PBP方法可以从小鼠ULF中分离出EV；分离得到的EV直径为62±19 nm，表达CD63、TSG101和HSP70蛋白。总之，在所研究的 4 种方法中，PBP 能够最好地保留分离的 EV 的活性，并且能够从少量样品中分离出 EV。简单的设置和较低的设备需求使得 PBP 成为临床和基础研究环境中快速评估 EV 和分离 EV 的最合适方法。
Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.
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影响因子：
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