[1]黄德发,张文娟,陈　杰,等.三种方法提取血清外泌体的比较研究[J].现代检验医学杂志,2021,36(06):139-142.[doi:10.3969/j.issn.1671-7414.2021.06.030]
　HUANG De-fa,ZHANG Wen-juan,CHEN Jie,et al.Comparative Study on Three Methods for Extraction of Serum Exosomes[J].Journal of Modern Laboratory Medicine,2021,36(06):139-142.[doi:10.3969/j.issn.1671-7414.2021.06.030]
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三种方法提取血清外泌体的比较研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第36卷
期数:
2021年06期
页码:
139-142
栏目:
研究简报·实验技术
出版日期:
2021-12-15

文章信息/Info

Title:
Comparative Study on Three Methods for Extraction of Serum Exosomes
文章编号:
1671-7414（2021）06-139-04
作者:
黄德发; 张文娟; 陈　杰; 等
（1. 赣南医学院第一临床医学院，江西赣州 341000；2.赣南医学院第一附属医院检验科，江西赣州 341000）
Author(s):
HUANG De-fa;  ZHANG Wen-juan;  CHEN Jie;  et al
（1. the First Clinical Medical College of Gannan Medical College , Jiangxi Ganzhou 341000, China; 2. Department of Laboratory Medicine, the First Affiliated Hospital of Gannan Medical College, Jiangxi Ganzhou 341000, China）
关键词:
外泌体; 超速离心法; 沉淀法; 尺寸排阻色谱法
分类号:
R446
DOI:
10.3969/j.issn.1671-7414.2021.06.030
文献标志码:
A
摘要:
目的　比较超速离心法、沉淀法和尺寸排阻色谱法提取血清外泌体的得率和纯度。方法　选取8例健康体检受试者血清，用三种方法提取外泌体，采用纳米流式检测仪、透射电子显微镜和Pierce BCA 蛋白检测试剂盒分别检测外泌体颗粒数、粒径分布、形态特征和蛋白浓度；分析外泌体颗粒数。外泌体颗粒数和蛋白总量比值、Triton X-100处理前后颗粒数变化来评估外泌体提取的得率和纯度。结果　超速离心法、沉淀法和尺寸排阻色谱法提取外泌体粒径均分布在40~150 nm；透射电子显微镜下均能观察到呈明显茶托状的双层膜结构，但沉淀法提取的外泌体混有聚乙二醇聚合物及蛋白大颗粒；蛋白质印迹结果显示三种方法提取的外泌体均表达外泌体标记蛋白；沉淀法提取的血清外泌体颗粒数高于超速离心法和尺寸排阻色谱法[(1.11±0.07)×1011 particley/ml vs (2.54±0.04)×1010particley/ml, (7.51±0.89)×1010particley/ml]，差异均有统计学意义（P<0.05），尺寸排阻色谱法提取外泌体颗粒数高于超速离心法（P<0.001） ；尺寸排阻色谱法提取外泌体纯度高于超速离心法和沉淀法[(5.66±0.45)×107particles/μg vs (7.65±0.42)×106 particles/μg, (3.18±0.45)×106 particles/μg]，超速离心法提取外泌体纯度高于沉淀法，差异均有统计学意义（P<0.05）。结论　超速离心法、沉淀法和尺寸排阻色谱法均能提取出血清外泌体，超速离心法步骤繁琐,耗时较长，提取外泌体得率与纯度较低；沉淀法操作简单，耗时短，提取外泌体得率最高，但外泌体纯度最低；尺寸排阻色谱法操作简单，耗时短，提取外泌体的纯度最高；相比而言，尺寸排阻色谱法显然优于超速离心法和沉淀法。
Abstract:
Objective　To compare the efficiency and purity of ultracentrifugation，precipitation and size-exclusion chromatography for extracting exosomes from serum. Methods　Three methods were used to extract serum exosomes. The number of exosomal particles, particle size distribution, morphological characteristics and protein concentration were detected by using nano flow detector, transmission electron microscope and Pierce BCA protein detection kit. Analyzed the number of exosomal particles, the ratio of the number of exosomal particles to total protein and the changes in the number of exosomal particles before or after Triton X-100 treatment to evaluate the yield and purity of exosomes extraction. Results　The particle size of the exosomes which had been extracted by ultracentrifugation, precipitation and size-exclusion chromatography were all distributed in the range of 40～150 nm, and the bilayer membrane with obvious saucer-like structure could be observed under transmission electron microscope, but the exosomes extracted by precipitation were mixed with polyethylene glycol polymers and large protein particles. The results of Western blot showed that the exosomes extracted by all three methods had expressed the tag proteins. The number of exosomal particles extracted by precipitation was higher than that of ultracentrifugation and size-exclusion chromatography [（1.11±0.07）×1011 particles/ml vs（2.54±0.04）×1010 particles/ml, （7.51±0.89）×1010 particles/ml]，the differences were statistically significant(all P<0.05), and the number of exosomal particles extracted by size-exclusion chromatography was higher than that of ultracentrifugation (P<0.001). The purity of exosomes extracted by size-exclusion chromatography was higher than that of ultracentrifugation and precipitation [（5.66±0.45）×107 particles/μg vs（7.65±0.42）×106 particles/μg，（3.18±0.45）×106 particles/μg], and the purity of exosomes extracted by ultracentrifugation was higher than that of precipitation, the differences were statistically significant(P<0.05). Conclusion　Ultracentrifugation, precipitation and size-exclusion chromatography can extract the exosomes of Xueqing. Ultracentrifugation is cumbersome, time-consuming and the yield and purity of exosomes are low. The precipitation method is simple and time consuming, and the extraction yield of exosomes is the lowest, but the purity of exosomes its the lowest. Size exclusion chromatography is simple and time-consuming, and the purity of secretes is the highest. In contrast, size-exclusion chromatography is obviously better than ultracentrifugation and precipitation.

参考文献/References:

[1] PEGTEL D M, GOULD S J. Exosomes[J]. AnnualReview of Biochemistry, 2019, 88(1):487-514
[2] JEPPESEN D K, FENIX A M, FRANKLIN J L, et al.Reassessment of exosome composition[J]. Cell, 2019,177(2): 428-445，e18.
[3] LATIFKAR A, HUR Y H, SANCHEZ J C, et al. Newinsights into extracellular vesicle biogenesis andfunction[J]. Journal of Cell Science, 2019, 132(13):jcs222406.
[4] XIAO Yongwei, ZHONG Jianing, ZHONG Baoyuan,et al. Exosomes as potential sources of biomarkers incolorectal cancer[J]. Cancer Letters, 2020, 476: 13-22.
[5] 蒲双双, 李金星. 外泌体在疾病实验诊断和临床治疗中的研究进展[J].现代检验医学杂志, 2018, 33 (4):160-164.PU Shuangshuang, LI Jinxing. Research progressof exosome in the laboratory diagnosis and clinictreatment of disease [J]. Journal of Modern LaboratoryMedicine, 2018,33(4):160-164.
[6] LI Pin, KASLAN M, LEE S H, et al. Progress inexosome isolation techniques[J]. Theranostics, 2017,7(3): 789-804.
[7] YAMAUCHI M, SHIMIZU K, RAHMAN M, et al.Efficient method for isolation of exosomes from rawbovine milk[J]. Drug Development and IndustrialPharmacy, 2019, 45(3): 359-364.
[8] BU Hongchang, HE Dinggeng, HE Xiaoxiao, et al.Exosomes: isolation, analysis, and applications incancer detection and therapy[J]. Chembiochem, 2019,20(4): 451-461.
[9] TIAN Ye, GONG Manfei, HU Yunyun, et al. Qualityand efficiency assessment of six extracellular vesicleisolation methods by nano-flow cytometry[J]. Journalof Extracellular Vesicles, 2020, 9(1): 1697028.
[10] GARDINER C, DI VIZIO D, SAHOO S, et al.Techniques used for the isolation and characterizationof extracellular vesicles: results of a worldwidesurvey[J]. Journal of Extracellular Vesicles, 2016, 5(1):32945.
[11] XIAO Yongwei, ZHENG Lei, ZOU Xiaofeng, et al.Extracellular vesicles in type 2 diabetes mellitus: keyroles in pathogenesis, complications, and therapy[J].Journal of Extracellular Vesicles, 2019, 8(1): 1625677.
[12] 范维肖, 刁艳君, 马越云, 等. 超速离心法与QIAGEN膜亲和柱法提取前列腺癌细胞培养上清外泌体的方法学比较[J]. 现代检验医学杂志, 2019, 34 (3):6-9.FAN Weixiao, DIAO Yanjun, MA Yueyun, et al.Comparison of ultracentrifugation and membranebased-affinity column methods in exosome isolationfrom supernatants of prostate cancer cells [J]. Journalof Modern Laboratory Medicine, 2019,34(3):6-9.
[13] MONGUI?-TORTAJADA M, G?LVEZ-MONT?NC, BAYES-GENIS A, et al. Extracellular vesicleisolation methods: rising impact of size-exclusionchromatography[J]. Cellular and Molecular LifeSciences, 2019, 76(12): 2369-2382.
[14] MONGUI?-TORTAJADA M, MOR?N-FONT M,G?MEZ-VALERO A, et al. Extracellular-vesicleisolation from different biological fluids by Size-Exclusion chromatography[J]. Current Protocols inStem Cell Biology, 2019, 49(1): e82.
[15] KONOSHENKO M Y, LEKCHNOV E A, VLASSOVA V, et al. Isolation of extracellular vesicles: generalmethodologies and latest trends[J]. BioMed ResearchInternational, 2018, 2018: 8545347.
[16] RIDER M A, HURWITZ S N, MECKES D J.ExtraPEG: A polyethylene Glycol-Based methodfor enrichment of extracellular vesicles[J]. ScientificReports, 2016, 6: 23978.
[17] GUAN Sheng, YU Hailong, YAN Guoquan, et al.Characterization of urinary exosomes purified with sizeexclusion chromatography and ultracentrifugation[J].Journal of Proteome Research, 2020, 19(6): 2217-2225.

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备注/Memo

备注/Memo:
基金项目：国家自然科学基金（81702580）；赣南医学院校创新课题（YC2020-X012）。作者简介：黄德发（1994-），男，在读硕士研究生，检验技师，主要从事外泌体在癌症诊断中应用的研究，E-mail：aa3716340@163.com。通讯作者：钟田雨（1982-），男，教授，检验科主任，主要从事外泌体在癌症诊断中应用的研究，E-mail：zhongtianyu@gmail.com。
更新日期/Last Update: 1900-01-01