【摘要】 目的比较四种方法所提取的人脐带间充质干细胞细胞外囊泡（EV）的效率。方法收集人脐带间充质干细胞的培养基上清液,通过差速离心法（方法A）、超滤结合差速离心法（方法B）、超滤结合聚乙二醇沉淀法（方法C）、超滤结合水两相系统法（方法D）提取EV。利用二辛可宁酸（BCA）蛋白浓度测定试剂盒检测各组EV的总蛋白浓度, Western blot法检测EV表面标志物凋亡关联基因2相互作用蛋白X（Alix）、 CD9与阴性对照钙连蛋白（calnexin）,透射电镜检测EV的形态,纳米颗粒跟踪分析检测EV的粒径分布与颗粒数。结果以上四种方法提取的EV蛋白含量分别为（1.92±1.77）μg/μL、（18.10±1.07）μg/μL、（6.33±1.02）μg/μL、（36.48±23.13）μg/μL。A、 B、 C三种方法所分离的EV的CD9、 Alix均呈阳性,其中方法C的表达量最低;阴性蛋白calnexin均呈阴性。方法D所得EV检测CD9、 Alix、 calnexin均呈现阴性。A、 B、 C三种方法提取的EV颗粒数分别为0.85×10¹¹/mL、 0.63×10¹¹/mL、 1.83×10¹¹/mL,且粒径分布均符合细胞外囊泡的粒径范围。透射电镜检测A、 B、 C三种方法所提EV均有典型的杯托状膜结构。结论超滤结合差速离心法可应用于对EV需求量大的实验。若EV需求量小,则建议直接使用差速离心法,降低EV破碎的风险。更多还原

【Abstract】 Objective To compare the efficiency of four methods for extracting extracellular vesicles（EVs） from human umbilical cord mesenchymal stem cells（hUCMSCs）. Methods EVs were isolated from the conditioned medium of hUCMSCs by ultracentrifugation（group A）, or ultrafiltration combined with ultracentrifugation（group B）, or ultrafiltration combined with polyethylene glycol precipitation（group C）, or ultrafiltration combined with aqueous two phase system（group D）. The total protein concentration of EVs in each group was determined by BCA method. The expression of Alix, CD9, and calnexin were detected by Western blotting. The morphology of EVs was analyzed by transmission electron microscopy. The particle size distribution and particle concentration of EVs were measured by nanoparticle tracking analysis. Results The total protein concentrations of EVs extracted by the above four methods were（1.92±1.77） μg/μL,（18.1±1.07） μg/μL,（6.33±1.02） μg/μL,（36.48±23.13） μg/μL from group A to D respectively. We observed the expression of CD9 and Alix, but not calnexin, in EVs from group A, B and C. However, the expression levels of CD9 and Alix were lowest in group C. In addition, the expression of CD9, Alix and calnexin were undetectable in EVs from group D. The particle concentrations of EVs in group A, B and C were 0.85×10¹¹ particles/mL, 0.63×10¹¹ particles/mL, 1.83×10¹¹ particles/mL, respectively. Meanwhile, the particle distributions were all within the size range of EVs. We also observed the typical saucer-like membrane structure in EVs from group A, B and C. Conclusion The method of ultrafiltration combined with ultracentrifugation could be applied to the experiments demanding large amounts of EVs. The method of ultracentrifugation is recommended for the extraction of little amounts of EVs due to the lower risk of EV fragmentation.更多还原