人尿液中exosomes的分离及鉴定

目的分离人尿液中的exosomes,并对其超微形态学和免疫特性进行鉴定。方法以超速离心法分离健康志愿者混合尿液中的exosomes,通过负染电镜和免疫电镜技术对exosomes进行鉴定,运用1-D SDS-PAGE对其蛋白质组分进行分离。结果 4℃,17 000×g离心15 min后的上清液于4℃,200 000×g离心1 h可得到纯度较高的exosomes。负染电镜可见exosomes为扁平或球形小体,直径主要在60 nm左右;免疫电镜显示,黑色胶体金颗粒均匀分布在exosomes球形体表面,因其具有很高的电子密度而呈现为清晰可辨的黑点;1-D SDS-PAGE分析显示,exosomes高丰度条带主要分布于相对分子质量90 000～110 000之间,且出现了数条清晰的、相对分子质量在30 000～60 000之间的条带,小于30 000的条带明显减弱。结论已建立了从人尿液中分离exosomes的方法,为进一步研究潜在的疾病相关生物标志物奠定了基础。

Isolation and Identification of Exosomes in Human Urine

Objective To isolate exosomes from human urine and identify their ultrastructural morphology and immune characteristics.Methods Exosomes were isolated from the mixed urine of healthy volunteers by ultracentrifugation and identified by electron microscopy with negative staining and immunoelectron microscopy,from which protein component was separated by 1-D SDS-PAGE.Results The exosmoes with high purity was obtained by further centrifugation of the supernatant of urine,collected after centrifugation at 4℃,17 000 × g for 15 min,at 4℃,200 000 × g for 1 h.Negative staining under electron microscope showed that the exosomes were flat or spherical corpuscula,in a diameter of about 60 nm.Colloidal gold particles were distributed evenly on the surface of exosomes,which were shown as clear black spots due to high electron density under immunoelectron microscope.1-D SDS-PAGE showed a majority of exosome bands with high abundance were 90 000 ～ 110 000,several clear bands with relative molecular masses of 30 000 ～ 60 000,and few bands with relative molecular masses of less than 30 000.Conclusion A method for isolation of exosomes from human urine was developed,which laid a foundation of further study on potential biomarkers of diseases.