2种不同牛乳外泌体提取方法的比较研究

Comparative study of 2 kinds of different extraction methods of bovine milk exosomes

中文关键词:  牛乳  外泌体  提取  鉴定

英文关键词:bovine milk  exosomes  extraction  identification

中文摘要:

      目的 比较研究2种不同的牛乳外泌体提取方法。方法 分别采用超速离心法和蔗糖密度梯度离心法从牛乳中提取外泌体, 利用透射电镜、动态光散射仪、纳米颗粒追踪仪、免疫印迹等技术对牛乳外泌体进行全面的鉴定表征。结果 利用上述2种方法提取获得的牛乳外泌体呈现双层膜的杯托状结构, 总体粒径分布在30~200 nm之间且外泌体中常见的特异标记蛋白CD9、CD81、TSG101均存在, 不存在污染蛋白Calnexin。结论 超速离心法与蔗糖密度梯度离心法均可从牛乳中提取获得结构完整的外泌体, 与超速离心法相比, 通过蔗糖密度梯度离心法可获取杂质较少、粒径分布更集中的牛乳外泌体, 但该法耗时长、外泌体浓度较低。

英文摘要:

      Objective To compare and study 2 kinds of different methods of bovine milk exosomes. Methods The ultracentrifugation method and sucrose density gradient centrifugation method were used to extract exosomes from bovine milk. Transmission electron microscopy, dynamic light scattering, nanoparticle tracking and immunoblotting were used to comprehensively identify and characterize bovine milk exosomes. Results The results showed that the bovine milk exosomes extracted by the above 2 kinds of methods all had cup-like structures with double-layer membranes, and their overall particle size distribution was between 30-200 nm. The specific marker proteins CD9, CD81 and TSG101 were present, and there was no contamination protein Calnexin. Conclusion Both the ultracentrifugation method and the sucrose density gradient centrifugation method can be used to extract complete structural exosomes from bovine milk. Compared with the ultracentrifugation method, the bovine milk exosomes obtained by the sucrose density gradient centrifugation method have fewer impurities and more concentrated particle size distribution, but it takes a long time and the concentration of exosomes is lower.