马奶和酸马奶来源外秘体的分离及其对RAW264.7细胞细胞因子的作用

Isolation of mare's milk & koumiss-derived exosomes and its effect on cytokines of RAW264.7

目的以马奶和酸马奶作为原料分离和鉴定外泌体,观察其对巨噬细胞株Raw264.7几种细胞因子的影响。方法用TEM观察不同方法分离的马奶和酸马奶外泌体的形态结构。运用Western blot检测外泌体膜特异性蛋白,对其进行鉴定。利用酶联免疫吸附(ELISA)法检测外泌体和Raw264.7细胞共培养后的肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-12p40以及IL-10等细胞因子的分泌水平。结果 TEM视野中观察到直径小于100 nm的具有清晰膜的茶托或凹半球的外泌体样结构。Western blot检测到分子量为28~29 kDa的外泌体膜特异性蛋白CD63的富集。共培养实验中发现,马奶外泌体能够促进Raw264.7细胞的促炎因子TNF-α和IL-12p40的分泌,而酸马奶外泌体则能够促进抗炎因子IL-10的分泌。结论马奶和酸马奶中大量存在外泌体,并具有不同的免疫调节作用。该研究结果为马奶和酸马奶的外泌体后续研究甚至作用机制研究提供了新的思路和理论基础。

Objective To isolate the exosomes from Mare’s milk and Koumiss and observe its effect on cytokines of Raw264. 7 cells.Methods（1）The size and morphostructure of exosomes will be observed by TEM.（2）The membrane-specific protein of exosomes will be detected by Western blot analysis.（3）The secretion level of TNF alpha,IL-12 p40 and IL-10 after Raw264. 7 cells co-incubated with exosomes will be tested by ELISA. Results（1）The exosome-like vesicles,which was less than 100 nm in diameter and with a clear membrane and also with saucer or concave hemisphere structure was observed in the field of TEM vision.（2）The 28 to 29 kDa membrane-specific protein CD63 was detected in Western blot analysis.（3）After co-incubation of exosomes and Raw264. 7 cells,the results showed that Mare’s milk-derived exosomes could promote secretion of proinflammatory factor TNF alpha and IL-12 p40 while Koumiss-derived exosomes could promote the secretion of anti-inflammatory factors IL-10. Conclusion The Mare’s milk and Koumiss-derived exosomes show different immune regulation effect. This research provides a novel thought and theoretical basis for the research of exosomal contents and the working mechanism of Mare’s milk and Koumiss.