Wei Huabing, Qian Xiaoqing, Xie Feng, Cui Daxiang
Department of Thoracic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
School of Biomedical Engineering, Shanghai Jiaotong University, Shanghai, China.
Ann Transl Med. 2021 May;9(10):882. doi: 10.21037/atm-21-2075.
Extracellular vesicles (EVs) is a group of heterogeneous cell-derived membrane structures, which is composed of a large number of exosomes released by cells, microbubbles (MVs) and apoptotic bodies. The formation of exocrine body is a process of fine regulation, which includes four stages: initiation, endocytosis, polycystic body formation and exocrine body secretion. Ultracentrifugation is currently the gold standard for external body separation; it includes a series of centrifugation steps at a rotation speed of 100,000 rpm or more to purify exocrine bodies from protein contaminants. Thus far, some separation methods, such as ultracentrifugation, polymer-based exosome separation kits and immune affinity-based isolation using antibodies against exosome surface proteins, have been used for tumor exosome isolation. It is not very clear which method is more suitable for the separation of serum exosomes from lung cancer patients.
Two methods for the extraction of exosomes from serum samples of lung cancer patients, namely, ultra-high speed centrifugation (Ultra-Exo) and precipitation (Prekit-Exo), were analyzed and compared. The biological morphologies of the extracted exosomes were studied by negative staining matter with transmission electron microscopy and cryo-electron microscopy. The particle size and the distribution were detected using nanoparticle tracking analysis (NTA).
Bio-transmission electron microscopy revealed that the morphologies of exosomes extracted by ultracentrifugation were superior to exosomes extracted with the Prekit-Exo kit. Ultracentrifugation was able to extract more exosomes compared to the Prekit-Exo kit. NTA showed that the exosomes obtained by ultra-high speed centrifugation had a smaller particle size compared to exosomes obtained by precipitation (30.4±26.8 150.3±6.8 nm, respectively). It is possible that the precipitant used in the precipitation kit was extracted with the exosomes, thereby causing the particle size to increase. Notably, the particle size of the exosomes extracted by the precipitation kit method showed a relatively narrow range in size. This could be due to the coating effect of the precipitation reagent, reducing the difference in the particle size of the exosomes.
Exosomes collected from the serum of lung cancer patients using the two extraction methods differed in morphology and numbers, with the ultracentrifugation method being superior to the precipitation method.
细胞外囊泡（EVs）是一组异质性的细胞来源的膜结构，由细胞释放的大量外泌体、微泡（MVs）和凋亡小体组成。外泌体的形成是一个精细调控的过程，包括起始、内吞、多囊泡体形成和外泌体分泌四个阶段。超速离心是目前外泌体分离的金标准；它包括一系列以100,000转/分钟或更高转速的离心步骤，以从蛋白质污染物中纯化外泌体。迄今为止，一些分离方法，如超速离心、基于聚合物的外泌体分离试剂盒以及使用针对外泌体表面蛋白的抗体进行免疫亲和分离，已被用于肿瘤外泌体的分离。目前尚不清楚哪种方法更适合从肺癌患者血清中分离外泌体。
分析并比较了两种从肺癌患者血清样本中提取外泌体的方法，即超速离心法（Ultra-Exo）和沉淀法（Prekit-Exo）。通过透射电子显微镜和冷冻电子显微镜的负染物质研究提取的外泌体的生物学形态。使用纳米颗粒跟踪分析（NTA）检测颗粒大小和分布。
生物透射电子显微镜显示，超速离心提取的外泌体形态优于Prekit-Exo试剂盒提取的外泌体。与Prekit-Exo试剂盒相比，超速离心能够提取更多的外泌体。NTA显示，超速离心获得的外泌体颗粒大小比沉淀法获得的外泌体小（分别为30.4±26.8和150.3±6.8 nm）。沉淀试剂盒中使用的沉淀剂可能与外泌体一起被提取出来，从而导致颗粒大小增加。值得注意的是，沉淀试剂盒法提取的外泌体颗粒大小范围相对较窄。这可能是由于沉淀试剂的包被作用，减少了外泌体颗粒大小的差异。
使用两种提取方法从肺癌患者血清中收集的外泌体在形态和数量上存在差异，超速离心法优于沉淀法。
