一种新型血清外泌体分离方法的构建及其在乳腺癌微RNA检测中的应用

Establishment of a novel method for isolation of serum exosome and its application in the detection of microRNA in breast cancer

目的 :构建一种采用聚乙二醇(polyethylene glycol,PEG)富集血清中外泌体并进行分离的方法。方法:分别采用PEG8000、商品化EXOQuick试剂盒以及超速离心法富集血清外泌体。采用透射电子显微镜和Nano Sight NS90法对分离得到的外泌体的粒径进行检测。蛋白质印迹法比较3种方法获得的血清外泌体表面标志物CD9表达的丰度;实时荧光定量PCR法检测乳腺癌、乳腺良性病变及健康志愿者血清和血清外泌体中微RNA-21(micro RNA-21,mi R-21)的表达趋势。结果 :本研究构建获得的PEG分离外泌体的方法,在粒径分布和外泌体表面标志物CD9表达丰度方面,优于超速离心法,与商品化的EXOQuick试剂盒效果相当。PEG法富集获得的乳腺癌患者血清外泌体中mi R-21的表达水平较乳腺良性病变明显上调(P=0.024),上调倍数为2.68倍;而直接从血清中检测mi R-21在乳腺疾病患者血清中表达水平的结果显示,乳腺癌患者和乳腺良性病变患者基本没有变化(P=0.373))。结论 :本研究构建的外泌体富集试剂及其方法,操作简便、耗时短、成本低。与直接检测血清中mi RNA的表达水平相比,可明显增加检测效率。

Objective： To establish a method for the isolation exosome from serum by polyethylene glycol （PEG）. Methods： The ultracentrifugation, commercial EXOQuick precipitation kit and PEG8000 precipitation method were used to isolate exosomes from blood serum of the patients with breast cancer and benign breast lesions and the healthy individuals, respectively. The morphology and particle size of exosomes were measured by transmission electron microscopy and NanoSight NS90 method, respectively. The expression level of the surface marker CD9 in serum exosomes was determined by Western blotting. The expression level of microRNA-21 （miR-21） in serum samples and serum exosomes was detected by real-time fluorescent quantitative PCR.Results： A method of isolating exosomes by PEG was established successfully, in terms of particle size distribution and CD9 concentration on the surface of exosomes, the performance of PEGS000 precipitation method was superior to ultracentrifugation, and equivalent to the commercial EXOQuick kit. Compared with breast benign lesions, the level of miR-21 was up- regulated by 2.68 fold （P=0.024） in serum exosomes isolated from malignant breast cancer patients by using PEGS000 precipitation method. However, the miR-21 expression level was almost equal in serum samples from the patients with breast benign lesions and breast cancer （P=0.373）.Conclusion： PEG8000 precipitation method for enriching exosomes is easy to operate, short of time and low in cost. The detection efficiency of miRNA expression in exosomes can be increased significantly than that in serum samples.