Zhen Ke, Wei Xiaojuan, Zhi Zelun, Zhang Shuyan, Cui Liujuan, Li Yue, Chen Xia, Yao Jing, Zhang Hongchao
The First Clinical Medical College, Hebei North University, Zhangjiakou 075132, China.
Department of Cardiovascular Surgery, Air Force Medical Center, People's Liberation Army, Beijing 100048, China.
Life (Basel). 2022 Nov 21;12(11):1942. doi: 10.3390/life12111942.
Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.
细胞外囊泡常见于人体体液中，能够反映人体当前的生理状况，并作为疾病的生物标志物。分离得到的细胞外囊泡的质量有助于各种伴有高脂血症疾病的早期诊断。然而，尚无关于哪种特殊方法适合从高脂血症患者血浆中分离细胞外囊泡的报道。因此，本研究比较了三种基于研究的不同细胞外囊泡分离方法，即超速离心法（UC）、聚乙二醇（PEG）沉淀法和尺寸排阻色谱法（SEC），并确定哪种方法最为有效。我们选取了12例临床诊断为高脂血症患者的血样，并用三种方法分离血浆来源的细胞外囊泡。使用透射电子显微镜观察分离得到的细胞外囊泡的形态，同时通过不对称流场-流分馏和多角度光散射检测其浓度。通过蛋白质免疫印迹法鉴定标志物蛋白，并用银染法评估蛋白质组成。二者均用于确定细胞外囊泡样品中的污染物。结果表明，这三种方法均可成功用于细胞外囊泡的分离。通过超速离心法分离得到的细胞外囊泡尺寸较大，但产量低得多。虽然通过聚乙二醇沉淀法分离得到的细胞外囊泡产量有了很大提高，但污染增加了。在这三种方法中，只有通过尺寸排阻色谱法分离得到的细胞外囊泡具有高产率和低污染的特点。因此，我们的数据表明，尺寸排阻色谱法是从高脂血症患者血浆中分离细胞外囊泡的更理想方法。
