人脐血血浆外泌体提取方法的比较

Comparison of the extraction methods of plasma exosomes from human umbilical cord blood

摘要:

为探寻高效且稳定的提取人脐血血浆外泌体的方法，利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇（polyethylene glycol，PEG）沉淀法提取人脐血血浆外泌体，并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小；聚氰基丙烯酸正丁酯（bicinchoninic acid，BCA）法测定外泌体蛋白总量；Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130（高尔基标志蛋白）的表达。结果表明，与提取外泌体的“金标准”，即超高速离心法相比，蔗糖垫密度梯度离心法稳定性好，获取的外泌体粒径较均一，但操作较复杂，耗时长；改良超速离心法操作较简单，纯度较高；PEG沉淀法提取的外泌体蛋白量最高，操作时间最短，但杂质较多。结果表明，4种方法均能从人脐血血浆中获取外泌体，但在操作时间、纯度、提取量等方面存在一定差异。因此，应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。

Abstract:

We compared ultracentrifugation, sucrose gradient centrifugation, improved ultracentrifugation, and polyethylene glycol (PEG) precipitation in the extraction of plasma exosomes from human umbilical cord blood, aiming at screening out a stable and efficient method. The morphology, structure, and size of exosomes were observed based on transmission electron microscopy and dynamic light scattering. Total protein content of exosomes was determined by bicinchoninic acid (BCA) assay, and the expression of exosome markers CD63 and HSP70 and exosome negative marker GM130 (Golgi marker) by Western blotting. Results showed that sucrose gradient centrifugation was more stable and yielded exosomes of uniform particle size compared with ultracentrifugation which had been considered as the "gold standard" for exosome extraction. However, sucrose gradient centrifugation had the limitations of complex operation and time-intensiveness. The improved ultracentrifugation featured ease of implementation and the extracted exosomes were of high purity. PEG precipitation extracted the most exosomes in a shorter timeframe, but the purity of the exosomes was low. In conclusion, all the four methods can separate exosomes from human umbilical cord blood plasma, but they are different in operation time, product purity, and product content. Therefore, the method for extracting plasma exosomes from human umbilical cord blood should be selected based on the experimental purpose and specific requirements.