Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.
Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.
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DOI：
10.1186/s12967-016-0935-4
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发表时间：
2016-06-18
影响因子：
7.4
通讯作者：
Colas E
中科院分区：
文献类型：
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作者：
Campoy I;Lanau L;Altadill T;Sequeiros T;Cabrera S;Cubo-Abert M;Pérez-Benavente A;Garcia A;Borrós S;Santamaria A;Ponce J;Matias-Guiu X;Reventós J;Gil-Moreno A;Rigau M;Colas E
子宫抽吸物用于子宫内膜疾病的诊断过程，但如果其复杂的环境被简化，可能会出现进一步的应用。从子宫抽吸物中分离的外泌体样囊泡可能成为生物标志物的有吸引力的来源，但需要标准化分离方案。本研究的目的是确定是否存在外泌体样囊泡的子宫抽吸液的流体部分，并比较协议，其分离，表征和分析。我们收集了39名患有良性妇科疾病的绝经前妇女的子宫抽吸物。将其中27份抽吸物的液体部分合并并分成等体积，以评价三种基于差速离心的程序：（1）标准方案，（2）过滤方案，和（3）蔗糖垫方案。通过电子显微镜、纳米颗粒跟踪分析和免疫印迹来评估分离的囊泡的表征。特别是对于RNA材料，我们评估了超声处理和RNA酶A处理在方案不同步骤的效果。我们最终证实了所选方法在非合并样本中的有效性。所有方案都可用于分离外泌体样囊泡。然而，标准程序是从子宫抽吸物中分离外泌体样囊泡的最佳执行方案：纳米颗粒跟踪分析显示具有135 ± 5 nm模式的较高浓度的囊泡，并且免疫印迹显示外泌体相关标志物（CD 9、CD 63和CD 81）的较高表达，从而验证了这种类型囊泡的富集。在没有超声处理和用RNaseA消化外源核酸的情况下成功地提取了外泌体样囊泡中包含的RNA，从而允许通过实时qPCR分析特异性内载物。我们证实了子宫抽吸液中存在外泌体样囊泡。通过差速离心成功地分离它们，为进一步分析提供足够的蛋白质组和转录组材料。标准方案是执行最好的程序，因为其他两个测试的方案既不改善外泌体样囊泡的产率也不改善外泌体样囊泡的纯度。本研究有助于为未来的比较研究奠定基础，以促进妇科生物标志物的研究领域。本文的在线版本（doi：10.1186/s12967-016-0935-4）包含补充材料，可供授权用户使用。
Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology. The online version of this article (doi:10.1186/s12967-016-0935-4) contains supplementary material, which is available to authorized users.