Charge-based precipitation of extracellular vesicles.
Charge-based precipitation of extracellular vesicles.
复制标题
DOI：
10.3892/ijmm.2016.2759
复制
发表时间：
2016-11
影响因子：
5.4
通讯作者：
Camussi G
中科院分区：
文献类型：
--
作者：
Deregibus MC;Figliolini F;D'Antico S;Manzini PM;Pasquino C;De Lena M;Tetta C;Brizzi MF;Camussi G
囊泡介导的细胞间通讯在许多生物过程中似乎是至关重要的。细胞外小泡(EV)从健康和疾病细胞中释放出来，参与生物活性分子的交换网络。由于存在于生物体液中的EV带有起源细胞的特征，它们是正在进行的生理或病理过程的潜在生物标记物。尽管近年来有关EV生物学的知识不断积累，但EV的纯化技术仍然是一个挑战，所有描述的方法都有一些优点和缺点。在本研究中，我们描述了一种基于EV从生物液和细胞上清液中电荷沉淀的方法，并与被认为是EV纯化的金标准的差速超速离心法进行了比较。ζ电势分析表明，电动汽车具有负电荷，允许与带正电荷的分子相互作用，如鱼精蛋白。鱼精蛋白可以诱导血清、唾液和细胞培养上清液中的EV沉淀，而不需要超速离心法。当鱼精蛋白(P)在聚乙二醇35000Da(P/PEG沉淀)存在的情况下进行沉淀时，EV的再悬浮被促进。用NanoSight分析评估沉淀EVS的回收比超速离心法更有效。在电子显微镜下，两种方法后的EVS大小相似，CD63、CD9和CD81外体标记在P/PEG化的EVS中的表达表明外切体中有丰富的EVS。P/PEG沉淀的EVS的RNA回收率与超速离心法分离的EVS相似。此外，通过角质形成细胞对伤口愈合和肾小管上皮细胞增殖的诱导，P/PEG沉淀的EVS在体外保持了生物活性。综上所述，电荷沉淀法具有操作简单、不需要昂贵设备的优点，可用于从小生物样品中高效分离电动汽车。
Vesicular-mediated communication between cells appears critical in many biological processes. Extracellular vesicles (EVs) released from healthy and diseased cells are involved in a network of exchange of biologically active molecules. Since EVs present in biological fluids carry the signature of the cell of origin, they are potential biomarkers for ongoing physiological or pathological processes. Despite the knowledge on EV biology accrued in recent years, techniques of EV purification remain a challenge and all the described methods have some advantages and disadvantages. In the present study, we described a method based on charge precipitation of EVs from biological fluids and from cell supernatants in comparison with the differential ultracentrifugation, which is considered the gold standard for EV purification. The analysis of ζ-potential revealed that EVs have a negative charge that allows the interaction with a positively charged molecule, such as protamine. Protamine was shown to induce EV precipitation from serum and saliva and from cell culture media without the need for ultracentrifugation. EV resuspension was facilitated when protamine (P) precipitation was performed in the presence of PEG 35,000 Da (P/PEG precipitation). The recovery of precipitated EVs evaluated by NanoSight analysis was more efficient than that obtained by ultracentrifugation. By electron microscopy the size of EVs was similar after both methods were used, and the expression of CD63, CD9 and CD81 exosomal markers in the P/PEG-precipitated EVs indicated an enrichment in exosomes. The RNA recovery of P/PEG-precipitated EVs was similar to that of EVs isolated by ultracentrifugation. In addition, P/PEG-precipitated EVs retained the biological activity in vitro as observed by the induction of wound closure by keratinocytes and of proliferation of tubular epithelial cells. In conclusion, charge-based precipitation of EVs has the merit of simplicity and avoids the requirement of expensive equipments and may be used for the efficient isolation of EVs from small biological samples.
登录
查看更多内容
影响因子：
5.4
作者：
Szatanek R;Baran J;Siedlar M;Baj-Krzyworzeka M
通讯作者：
Baj-Krzyworzeka M
影响因子：
4
作者：
Momen-Heravi F;Balaj L;Alian S;Trachtenberg AJ;Hochberg FH;Skog J;Kuo WP
通讯作者：
Kuo WP
影响因子：
7.5
作者：
Quesenberry PJ;Aliotta J;Deregibus MC;Camussi G
通讯作者：
Camussi G
DOI：
10.1007/978-1-61779-068-3_15
发表时间：
2011-01-01
期刊：
SERUM/PLASMA PROTEOMICS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Taylor, Douglas D.;Zacharias, Wolfgang;Gercel-Taylor, Cicek
通讯作者：
Gercel-Taylor, Cicek
影响因子：
2.8
作者：
Rekker, Kadri;Saare, Merli;Peters, Maire
通讯作者：
Peters, Maire