目的:建立阴选纯化富集(NSPE)法,从血清中高效、快速提取外泌体,并对外泌体进行鉴定。方法:利用低密度脂蛋白受体与低密度脂蛋白(LDL)结合的原理,去除人血清中大量LDL干扰,再通过外泌体表面磷脂分子的磷酸盐基团与二氧化锆(ZrO2)双配位结合的原理富集外泌体。使用纳米流式细胞仪、透射电子显微镜来检测外泌体的粒径分布、颗粒浓度和形态特征,蛋白免疫印迹法检测外泌体表面标志性蛋白。使用基因本体论数据库和京都基因与基因组百科全书数据库对外泌体内部的microRNAs (miRNAs)进行基因通路分析。结果:该方法可在60 min从血清中有效提取外泌体。外泌体颗粒浓度为(2.94±0.3)×1010颗粒·mL-1,粒径大小为(88.07±2.94) nm,并呈现标志性蛋白TSG101+、CD9+。外泌体内部miRNAs与免疫、代谢过程等相关。结论:建立的NSPE法可从血清中高效、快速地提取外泌体,用于健康监测和医学检验。
Objective: To establish a method of negative selection purification-enrichment (NSPE) extract exosomes from serum efficiently and rapidly, and exosomes were identified. Methods: A large amount of low density lipoproteins (LDL) interference in human serum was removed based on the principle of combining the low density lipoprotein receptor with low density lipoprotein. Then, exosomes were enriched by the principle of bidentate binding between the phosphate groups of phospholipid molecules on the surface of exosomes and ZrO2. Nanoflow cytometry and transmission electron microscopy were used to detect the particle size distribution, particle concentration and morphological characteristics of exosomes, and western blotting was used to identify surface specific proteins expression of exosomes. MicroRNAs (miRNAs) in exosomes were analyzed for gene pathway using the Gene Ontology database and the Kyoto Encyclopedia of Genes and Genomes database. Results: The exosomes in serum were effectively extracted using the method here within 60 minutes. The concentration of exosome particles was about (2.94±0.3)×1010 particles·mL-1, and the particle size was (88.07±2.94) nm. The expressions of exosome surface specific proteins, TSG101+ and CD9+, were detected. MiRNAs derived from exosomes were related to immune process and metabolic process. Conclusion: The established NSPE method could be used for rapid and efficient extraction of exosomes from serum for health monitoring and medical examination.
[1] ZHANG Y, BI JY, HUANG JY, et al. Exosome: a review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications[J].Int J Nanomed, 2020, 2020(15):6917
[2] YANG XX, SUN C, WANG L, et al. New insight into isolation, identification techniques and medical applications of exosomes[J].J Control Release, 2019, 2019(308):119
[3] HU T, WOLFRAM J, SRIVASTAVA S. Extracellular vesicles in cancer detection: hopes and hypes[J].Trends Cancer, 2021,7(2):122
[4] KAMINSKA P, BUSZKA K, ZABEL M, et al. Liquid biopsy in melanoma: significance in diagnostics, prediction and treatment monitoring[J].Int J Mol Sci, 2021,22(18):9714
[5] LOGOZZI M, MIZZONI D, DI RR, et al. Plasmatic exosome number and size distinguish prostate cancer patients from healthy individuals: a prospective clinical study[J].Front Oncol, 2021,2021(11): 727317
[6] YIN L, LIU XT, SHAO XJ, et al. The role of exosomes in lung cancer metastasis and clinical applications: an updated review[J].J Transl Med, 2021, 19(1): 312
[7] BESTARD EJ, REEIGADA R, REYES J, et al. Fatty acid unsaturation degree of plasma exosomes in colorectal cancer patients: a promising biomarker[J].Int J Mol Sci, 2021, 22(10):5060
[8] RASTOGI S, SHARMA V, BHARTI PS, et al. The evolving landscape of exosomes in neurodegenerative diseases: exosomes characteristics and a promising role in early diagnosis[J].Int J Mol Sci,2021, 22(1):440
[9] HWANG S, YANG YM. Exosomal microRNAs as diagnostic and therapeutic biomarkers in non-malignant liver diseases[J].Arch Pharm Res, 2021, 44(6): 574
[10] YANG DB, ZHANG WH, ZHANG HY, et al. Progress, opportunity, and perspective on exosome isolation efforts for efficient exosome-based theranostics[J].Theranostics, 2020, 10(8): 3684
[11] SIMONSEN JB. What are we looking at? Extracellular vesicles, lipoproteins, or both?[J].Circ Res, 2017, 121(8): 920
[12] ZHOU HJ,TIAN RJ, YE ML, et al. Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis[J].Electrophoresis, 2007, 28(13): 2201
[13] WANG F, ZHANG XH, LIU YB, et al. Profiling metal oxides with lipids: magnetic liposomal nanoparticles displaying DNA and proteins[J].Angew Chem(Int Ed Engl), 2016, 55(39): 12063
[14] LANGEVIN SM, KUHNELL D, ORRASMAN MA, et al. Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum[J].RNA Biol, 2019, 16(1): 5
[15] CHEN J, JIAO ZG, MO JW, et al. Co-mparison of the variability of small extracellular vesicles derived from human liver cancer tissues and cultured from the tissue explants based on a simple enrichment method.[J].Stem Cell Rev Rep, 2021,18(3): 1067
[16] LIU T, ZHANG Q, ZHANG JK, et al. EVmiRNA: a database of miRNA profiling in extracellular vesicles[J].Nucleic Acids Res, 2019, 47(D1): D89
[17] GAO FY, JIAO FL, XIA CS, et al. A novel strategy for facile serum exosome isolation based on specific interactions between phospholipid bilayers and TiO2[J].Chem Sci, 2019, 10(6): 1579