人脐带间充质干细胞来源外泌体提取方法的比较

摘要

背景:近年来"无细胞"的干细胞治疗方法备受关注,但对于提取高质量外泌体的方法尚无确切定论.目的:采用3种方法提取人脐带间充质干细胞中的外泌体,筛选最佳提取外泌体的方法.方法:分别用Total Exosome Isolation试剂盒、Exo Quick试剂盒及差速超速离心法提取人脐带间充质干细胞来源的外泌体.使用透射电镜观察外泌体形态,BCA法进行蛋白质定量,western blot检测外泌体表面标志物CD9、CD81、CD63的表达.结果与结论:①3种方法均可以从培养基中提取到间充质干细胞来源的外泌体,并且在透射电镜下观察到圆形或椭圆形的膜性小囊泡;②差速超速离心法提取的外泌体中的蛋白浓度和纯度最高,Exo Quick 试剂盒法其次,Total Exosome Isolation试剂盒法得到的蛋白浓度最低,且3者比较差异有显著性意义(P < 0.05);③3种方法提取的外泌体均表达其表面特异性标志物 CD81、CD63和CD9,且在含有相同蛋白浓度的情况下,差速超速离心法提取的外泌体的表面标志物含量最高,Exo Quick 试剂盒法其次,Total Exosome Isolation试剂盒法提取的外泌体表面标志物含量较低;④差速超速离心法和Total Exosome Isolation试剂盒法的操作时间明显多于Exo Quick试剂盒法,差异有显著性意义(P < 0.05);⑤结果表明,尽管差速超速离心法耗时稍长,但是纯度相对较高,满足实验的基本需求,可作为一种合理的分离方法.%BACKGROUND: Cell-free stem cell therapy has been an issue of concern, but there is no conclusion on how to extract high-quality exosomes. OBJECTIVE: To extract exosomes from human umbilical cord mesenchymal stem cells by using three different methods, and then to screen the optimal method. METHODS: Exosomes were extracted from human umbilical cord mesenchymal stem cells by using the Total Exosome Isolation test kit, Exo Quick test kit and differential ultracentrifugation method, respectively. Then, transmission electron microscopy was used for morphological observations, BCA was utilized to quantify the protein, and western blot assay was applied to detect surface markers CD9, CD81 and CD63. RESULTS AND CONCLUSION: Extraction of exosomes was completed by all the three methods, and round or oval membranous vesicles were observed under the transmission electron microscope. The protein content and purity of exosomes was highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group, and there were significant differences among the three groups (P < 0.05). Under the same protein concentration, surface specific markers, CD81, CD63 and CD9, were expressed highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group. The operating time was significantly lower in the Exobiology Quick kit group compared with the other two groups (P < 0.05). To conclude, despite a longer operating time, the differential ultracentrifugation method is a rational method to extract enough exosomes with relative high purity.