两种乳腺癌患者血清来源的外泌体分离方法的比较

Comparison of ultracentrifugation and Prekit-exo Kit for the identification of exosomes extracted from Serum of breast cancer patients

目的 :探索乳腺癌患者血清来源的外泌体最佳纯化条件,并对所得的外泌体进行形态鉴定和浓度测定,以期获得较优的分离血清来源外泌体的纯化方法。方法 :收集患者血清,分别用超高速离心法(Ultra-exo)和沉淀试剂盒法(Prekit-exo)分离提取外泌体。生物透射电镜负染上样观察外泌体生物学形态,并用纳米激光粒度跟踪测量仪(NTA)检测其粒径大小和粒径分布,用western blot来进行蛋白表征鉴定。结果 :两种方法得到的外泌体,生物透射电镜下可见超高速离心获得的是具有典型半杯托状结构的外泌体,大小在30～200nm之间,而沉淀试剂盒法分离的产物不具有典型的外泌体形态,甚至有很多杂蛋白,且背景十分杂乱。此外,NTA显示超高速离心法得到的外泌体粒径分布均匀,在一个较窄的粒径范围(117.8±28.7nm)。而沉淀试剂盒法所得产物在粒径分布上呈现多段粒径区间,分布范围宽(289.5±103.3nm)。Western Blot结果显示,沉淀试剂盒法提取的外泌体杂蛋白多余超高速离心法。结论 :本实验结果表明,分离血清来源的外泌体,超高速离心法显然优于沉淀试剂盒法。

Objective To compare ultracentrifugation and Prekit-exo Kit in identifying the characteristics of exosomes extracted from Serum of breast cancer patients.Methods Serum samples were collected and extracted by ultra high speed centrifugation(Ultra-exo)and Precipitation Kit(Prekit-exo)respectively.The biological morphology of exosome was observed by negative coloring matter with transmission electron microscopy,and the particle size and the distribution were detected by nano particle size tracking instrument(NTA).Results Bio-transmission electron microscopy,revealed that the morphologys in the ultracentrifugation-extracted exosomes were better than those in the Prekit-exo-extracted exosomes.Results showed that the number of the ultracentrifugation-extracted exosomes was more than that from the Prekit-exo-extracted exosomes.Also NTA(Nanoparticle Tracking Analysis)shows the number of ultracentrifugation-extracted exosomes is more than number of Prekitexo-extracted exosomes.Western Blot results show that protein of exosomes obtained by Prekit-exo method are more and miscellaneous than Ultra-exo method.Conclusion Exosomes from Serum of breast cancer patients isolated by ultracentrifugation and precipitation are different in morphologys and numbers,and ultracentrifugation method is superior to precipitation method.