尿液外泌体提取优化及冷冻对外泌体RNA含量的影响  

Optimization for extraction of urine exosomes and effects of freezing on exosomal RNA content

作　　者：南阿妮 刁艳君 杨柳 马越云 苏明权 郝晓柯 NAN Ani;DIAO Yanjun;YANG Liu;MA Yueyun;SU Mingquan;HAO Xiaoke(Department of Clinical Laboratory, the First Affiliated Hospital of theFourth Military Medical University, Xi′an 710032, Shaanxi, China)

机构地区：[1]第四军医大学第一附属医院检验科

出　　处：《临床检验杂志》2019年第5期325-330,共6页Chinese Journal of Clinical Laboratory Science

摘　　要：目的对现有沉淀法提取尿液外泌体进行优化,并探讨不同的储存条件对尿液外泌体RNA含量的影响。方法分别用先浓缩后沉淀法和直接沉淀法提取人尿液标本中的外泌体,比较两种方法的分离效率和成本;采用ExoQuick-TCTM沉淀试剂盒提取外泌体;纳米粒子跟踪分析技术(NTA)检测外泌体浓度及粒径分布;动态光散射技术(DLS)检测外泌体电位;透射电镜(TEM)观察外泌体形态;western blot检测外泌体标记分子CD63及Alix蛋白的表达;另取10例临床尿液标本验证先浓缩后沉淀的提取方法,以证实优化方法的可靠性。实时荧光定量聚合酶链反应(qRT-PCR)检测20例前列腺癌患者尿液外泌体经过反复冻融(新鲜、1、3、5次)及9例前列腺癌患者尿液外泌体在-80℃冷冻不同时间(新鲜、1周、2周、1个月)时外泌体RNA标志物let-7c和PSA mRNA的表达量,并采用Wilcoxon秩和检验进行统计学分析。结果 NTA检测两种方法提取的外泌体粒径分布为30~150 nm,均为单峰;DLS结果显示,两种方法提取的外泌体电位均呈负值;透射电镜观察两种方法提取的外泌体大小较为一致,直径分布为30~150 nm;western blot检测证实优化方法存在外泌体标记分子CD63及Alix蛋白;10例尿液标本提取的外泌体浓度约为109~1011 particles/mL。外泌体经过5次冻融后let-7c和PSA mRNA含量明显下降,Z值分别为-1.69、-1.73(P均<0.05)。而外泌体在-80℃冷冻1个月后其RNA含量保持稳定。结论优化后的外泌体提取方法在保证外泌体浓度和质量的前提下极大地降低成本,分离得到的外泌体在-80℃短时间冷冻后其RNA含量保持稳定,但不能反复冻融超过5次。Objective To optimize the existing methods of isolation and purification for exosomes from urine and explore the effects of different storage conditions on the content of exosomal RNA in urine. Methods The exosomes in human urine samples were extracted by different precipitation method, i.e., precipitation following first concentrating and direct precipitation, respectively, and the separation efficiency and cost of the two methods were compared. ExoQuick-TCTM precipitation kit was used to extract exosomes. Nanoparticle tracking analysis technique(NTA) was used to detect the concentration and particle size distribution of exosome. Dynamic light scattering(DLS) was used to detect the potential of exosome. Transmission electron microscopy(TEM) was used to observe morphology of exosomes. western blot was used to analyze the exosomal marker molecules CD63 and Alix. The extraction method of the precipitation following first concentrating was used to verify the reliability of the optimized method in 10 clinical urine samples. Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) was used to detect the expression levels of exosomal RNA marker let-7 c and PSA mRNA in the urinary exosomes from 20 patients with prostate cancer after repeated freeze-thaw(0 [i.e., fresh], 1, 3 and 5 times) and 9 patients with prostate cancer frozen at-80 ℃ for different time(0 [i.e., fresh], 1, 2 and 4 weeks), and were statistically analyzed by Wilcoxon rank sum test for differences between the 2 groups. Results The size distribution of exosomes extracted by the two methods was 30 to 150 nm by NTA, both of which were displayed as single peaks. The results of DLS showed that the potentials of exosome extracted by the two methods were negative values. The size of the exosomes extracted by the two methods was consistent observed under TEM namely the diameter distribution was 30 to 150 nm. western blot analysis confirmed that CD63 and Alix, the exosome labeling molecules, existed in the optimized method. The concentration of e

关 键 词：尿液 外泌体 外泌体RNA 反复冻融 

分 类 号：R446.1[医药卫生—诊断学]