Research progress in extracellular vesicle detection technique and its clinical application

doi:10.3969/j.issn.1673-8640.2020.12.002

Abstract

Abstract:

Extracellular vesicles（EV）,a kind of subcellular components secreted by cells,carrying many signal molecules,participate in many physiological and pathological activities. The detection technique and analysis of its components are conducive to the specific analysis of diseases from the cellular,subcellular and molecular levels,which is of significance both to the mechanism research of disease and the clinical application of EV. This review summarizes the main detection technique of EV including the advantages and disadvantages. Meanwhile,its clinical applications will be evaluated and the brief outlook of its development will be made.

Key words: Extracellular vesicle, Biomarker, Detection technique

CLC Number:

R446.1

LIU Chunchen, LIN Huixian, ZHENG Lei. Research progress in extracellular vesicle detection technique and its clinical application[J]. Laboratory Medicine, 2020, 35(12): 1207-1212.

Figures/Tables 2

References 43

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分类	检测技术名称	优点	缺点
分离富集	超速离心法^[8]	操作简单,可用于大批量样本	所需设备昂贵,耗时,离心力过大或重复离心均可影响EV的完整性
	密度梯度离心法^[9]	分离纯度提高,可较好保持EV 活性	耗时,配制惰性梯度介质时需把握其渗透压
	尺寸排阻色谱法^[10]	分离纯度进一步提高,省时, 可较好保持EV活性	层析柱使用次数与上样量有限制,部分样本中的脂蛋白可能与EV共分离
	超滤法^[11]	操作简单,省时,可较好保持 EV活性	无法滤除小于滤膜孔径的杂质;样本需经前处理去除细胞等大的膜状结构,避免其经过滤膜时分解形成小囊泡,影响纯度与后续分析
	聚乙二醇沉淀法^[12]	试剂易得,操作较简单	耗时,易受其他疏水蛋白质干扰,影响EV后续蛋白功能分析
	免疫磁珠分选法^[13]	特异性好,纯度高	成本较高;受抗体制备技术限制;抗原表位可被激活或屏蔽,影响后续的功能分析
鉴定	透射电子显微镜^[14]、扫描电子显微镜^[15]、冷冻电子显微镜^[16]、原子力显微镜^[17]等	可用于EV形态学特征鉴定,透射电子显微镜可获取EV内部的结构特征,原子力显微镜可提供EV的三维表面图	对设备要求高,样本制备复杂,脱水固定以及染色可能影响EV活性,成本高
	纳米颗粒跟踪分析^[18]	可获取EV实时浓度与粒径分布信息	无法区分EV表型与来源,无法将EV与粒径相似的粒子相区分;不适合非均质样本,大颗粒光强信号易掩盖小颗粒信号
	免疫印迹法^[19]、ELISA^[20]	可对目标蛋白进行定性、半定量或定量分析,特异性强	只能对已知蛋白进行检测,受抗体制备技术限制,操作较繁琐、耗时

分类	检测技术名称	优点	缺点
分离富集	超速离心法^[8]	操作简单,可用于大批量样本	所需设备昂贵,耗时,离心力过大或重复离心均可影响EV的完整性
	密度梯度离心法^[9]	分离纯度提高,可较好保持EV 活性	耗时,配制惰性梯度介质时需把握其渗透压
	尺寸排阻色谱法^[10]	分离纯度进一步提高,省时, 可较好保持EV活性	层析柱使用次数与上样量有限制,部分样本中的脂蛋白可能与EV共分离
	超滤法^[11]	操作简单,省时,可较好保持 EV活性	无法滤除小于滤膜孔径的杂质;样本需经前处理去除细胞等大的膜状结构,避免其经过滤膜时分解形成小囊泡,影响纯度与后续分析
	聚乙二醇沉淀法^[12]	试剂易得,操作较简单	耗时,易受其他疏水蛋白质干扰,影响EV后续蛋白功能分析
	免疫磁珠分选法^[13]	特异性好,纯度高	成本较高;受抗体制备技术限制;抗原表位可被激活或屏蔽,影响后续的功能分析
鉴定	透射电子显微镜^[14]、扫描电子显微镜^[15]、冷冻电子显微镜^[16]、原子力显微镜^[17]等	可用于EV形态学特征鉴定,透射电子显微镜可获取EV内部的结构特征,原子力显微镜可提供EV的三维表面图	对设备要求高,样本制备复杂,脱水固定以及染色可能影响EV活性,成本高
	纳米颗粒跟踪分析^[18]	可获取EV实时浓度与粒径分布信息	无法区分EV表型与来源,无法将EV与粒径相似的粒子相区分;不适合非均质样本,大颗粒光强信号易掩盖小颗粒信号
	免疫印迹法^[19]、ELISA^[20]	可对目标蛋白进行定性、半定量或定量分析,特异性强	只能对已知蛋白进行检测,受抗体制备技术限制,操作较繁琐、耗时

内容物	检测技术名称	标志物	疾病类型	优点
核酸	基于多色荧光PCR的EV- lncRNA分析芯片^[24]	SLC9A3-AS1、PCAT6	肺癌	多重检测低水平EV的lncRNA,检测限为 10 拷贝/μL
	功能化金纳米荧光探针^[25]	miRNA-1246	乳腺癌	仅需40 μL样本;可用于区分乳腺癌患者与健康人（敏感性为100.0%,特异性为92.9%）
	免疫生物芯片^[26]	miRNA-21、TTF-1 mRNA	非小细胞肺癌	所需样本量少（30 μL）、耗时少（4 h）,可用于肺癌诊断
	NanoVilli芯片^[27]	EGFR、ROS1	非小细胞肺癌	EV富集仅需30 min,可联合实时定量数字PCR 完成mRNA分析;可用于肺癌ROS1重排及 EGFRT790M突变监测
蛋白质	外泌体双膜蛋白共表达检测平台^[34]	CD63、 EpCAM	乳腺癌	可用于区分乳腺癌来源与正常乳腺上皮来源的外泌体
	液滴数字ExoELISA^[35]	GPC-1	乳腺癌	检测限为10~17 mol,可用于靶外泌体的绝对计数
	荧光生物传感台^[36]	CD63、核仁素	白血病	灵敏度和特异性均较高,可检测浓度为100个/μL 的外泌体
	放大SAW传感器^[37]	CD63、 EpCAM		检测限达1.2×10³个/mL,用时短（30 min）
	Aptasensor^[38]	CD63、 MUC1	胃癌	可检测低至9.54×10²个/mL的靶外泌体,可应用于胃癌的诊断与预后评估
	Exodisc-B/P^[39]	CD9、CD81、CD63、 PSA、PSMA、 HSP90、EpCAM、 EGFR	前列腺癌等	样本用量为30 μL,EV捕获率高达75%,纯度与产量均高于传统超速离心法,可用于前列腺癌的辅助诊断及动物模型的肿瘤进展监测
脂质	基于激光镊子拉曼光谱的检测平台^[43]	胆固醇、磷脂等		不需标记,可保持外泌体活性,使单个外泌体水平的检测成为可能
	高通量质谱分析^[42]	PS（18：1/18：1）、 PS（18：0-18： 2）、LacCer（d18： 1/16：0）等	前列腺癌	LacCer（d18：1/16：0） /PS（18：1/18：1）比值与PS（18：0-18：2）/PS（18：1/18：1）比值联合检测区分前列腺癌与健康人的敏感性为93%、特异性为100%

内容物	检测技术名称	标志物	疾病类型	优点
核酸	基于多色荧光PCR的EV- lncRNA分析芯片^[24]	SLC9A3-AS1、PCAT6	肺癌	多重检测低水平EV的lncRNA,检测限为 10 拷贝/μL
	功能化金纳米荧光探针^[25]	miRNA-1246	乳腺癌	仅需40 μL样本;可用于区分乳腺癌患者与健康人（敏感性为100.0%,特异性为92.9%）
	免疫生物芯片^[26]	miRNA-21、TTF-1 mRNA	非小细胞肺癌	所需样本量少（30 μL）、耗时少（4 h）,可用于肺癌诊断
	NanoVilli芯片^[27]	EGFR、ROS1	非小细胞肺癌	EV富集仅需30 min,可联合实时定量数字PCR 完成mRNA分析;可用于肺癌ROS1重排及 EGFRT790M突变监测
蛋白质	外泌体双膜蛋白共表达检测平台^[34]	CD63、 EpCAM	乳腺癌	可用于区分乳腺癌来源与正常乳腺上皮来源的外泌体
	液滴数字ExoELISA^[35]	GPC-1	乳腺癌	检测限为10~17 mol,可用于靶外泌体的绝对计数
	荧光生物传感台^[36]	CD63、核仁素	白血病	灵敏度和特异性均较高,可检测浓度为100个/μL 的外泌体
	放大SAW传感器^[37]	CD63、 EpCAM		检测限达1.2×10³个/mL,用时短（30 min）
	Aptasensor^[38]	CD63、 MUC1	胃癌	可检测低至9.54×10²个/mL的靶外泌体,可应用于胃癌的诊断与预后评估
	Exodisc-B/P^[39]	CD9、CD81、CD63、 PSA、PSMA、 HSP90、EpCAM、 EGFR	前列腺癌等	样本用量为30 μL,EV捕获率高达75%,纯度与产量均高于传统超速离心法,可用于前列腺癌的辅助诊断及动物模型的肿瘤进展监测
脂质	基于激光镊子拉曼光谱的检测平台^[43]	胆固醇、磷脂等		不需标记,可保持外泌体活性,使单个外泌体水平的检测成为可能
	高通量质谱分析^[42]	PS（18：1/18：1）、 PS（18：0-18： 2）、LacCer（d18： 1/16：0）等	前列腺癌	LacCer（d18：1/16：0） /PS（18：1/18：1）比值与PS（18：0-18：2）/PS（18：1/18：1）比值联合检测区分前列腺癌与健康人的敏感性为93%、特异性为100%