主动脉夹层患者尿液来源外泌体不同分离方法的比较

目的 探讨分离主动脉夹层患者尿液来源外泌体的适宜方案。方法 收集主动脉夹层患者尿液，采用超速离心法、透析超滤法和沉淀试剂盒法分离提纯尿液中的外泌体。在生物透射电子显微镜下观察尿液外泌体的形态，用纳米颗粒跟踪分析仪检测其粒径大小、颗粒分布与浓度。结果 经过表征，3种方法获得的尿液外泌体特征如下：（1）超速离心法获得的尿液外泌体具有半杯托状或凹陷的半球形结构；粒径较大，分布较均匀；粒径范围较宽，为（236.4±46.5）nm；浓度较低，为（2.82±0.21）×10¹²个/mL；（2）透析超滤法获得的尿液外泌体形态较好，且数量多；粒径较小，分布较集中；粒径范围较窄，为（128.7±6.3）nm；浓度较大，为（2.16±0.15）×10¹⁴个/mL；（3）沉淀试剂盒法获得的产物在生物透射电子显微镜下未见明显的外泌体结构。结论 主动脉夹层患者采用透析超滤法分离获得的尿液外泌体浓度较高，在对外泌体的纯度要求不是特别高的情况下比较适宜。

Comparison of different separation protocols for exosomes derived from urine of patients with aortic dissection

Objective To explore a suitable separation protocol for exosomes derived from urine of patients with aortic dissection. Methods Urine samples were collected from the patients with aortic dissection. The exosomes were extracted and purified by ultracentrifugation, dialysis-ultrafiltration and precipitation kit methods. The biological morphology of exosomes was observed under transmission electron microscope. The particle size, distribution and concentration of the exosomes were detected by nanoparticle tracking analysis. Results The exosomes extracted with ultracentrifugation method had half cup-like or concave-hemispherical morphology; the particle size was large and well-distributed; the range of the particle size was wide with a mean value of (236.4±46.5) nm; and the concentration was low, being (2.82±0.21)×10¹² per 1 mL urine. The exosomes extracted by dialysis-ultrafiltration method had normal morphology and large quantity; the particle size was small and concentrated; the range of the particle size was narrow with a mean value of (128.7±6.3) nm; and the concentration was high, being (2.16±0.15)×10¹⁴ per 1 mL urine. There were no exosomes in the extractive by precipitation kit method under transmission electron microscope. Conclusion Dialysis-ultrafiltration method is a suitable method for extracting exosomes from urine of the aortic dissection patients and can yield a high concentration, while it is not suitable when exosomes of very high purity are required.