摘要目的 研究血浆中外泌体(exosomes)样小体对正常人巨噬细胞免疫调节作用的影响.方法 采用超速离心结合膜过滤的方法,从健康献血者血浆中分离纯化外泌体样小体.通过分离健康献血者外周血单个核细胞,贴壁培养并分离巨噬细胞.外泌体样小体与巨噬细胞共孵育后,采用流式细胞术、RT-PCR、Western blot法分别检测巨噬细胞胞质Ca2+、相关基因和蛋白的变化.结果 外泌体样小体与巨噬细胞孵育一段时间后,巨噬细胞胞质内Ca2+平均荧光强度上调;外泌体样小体对巨噬细胞的促炎因子如IL-1β、IL-6、TNF-α的表达有促进作用,在共培养2 h时,IL-1β、TNF-α基因表达达到高峰,分别比对照组表达上调0.85倍和1.69倍,而IL-6基因表达上调水平在共培养8 h时达到高峰,是对照组的3.7倍,而对抑炎因子IL-10的表达具有抑制作用;同时发现巨噬细胞表达Wnt5A受体Frizzled5,但是外泌体样小体对其基因表达没有明显影响;外泌体样小体能使钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)发生磷酸化.结论 血浆外泌体样小体能上调巨噬细胞炎症因子基因的表达,并且可能通过活化巨噬细胞的Wnt5A-Ca2+信号通路促进炎性细胞因子的分泌.

abstractsObjective To study the influence of human plasma exosomes-like vesicles on the regulatory function of macrophages. Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugation and ultrafiltration. Macrophages were derived from cultured human blood monocytes. The molecular markers of macrophages were assayed by FACS. After cultured with exosomes-like vesicles, the changes of macrophages cytoplasma Ca2 + , and related genes and proteins were assayed by FACS, RT-PCR and Western Blot, respectively. Results After cultured with exosomes-like vesicles, mean fluoresecent intensity (MFI) of macrophages cytoplasma Ca2+ was increased. The vesicles enhanced macrophages to express cytokines genes, the expression of IL-1 β and TNF-α genes being increased by 0.85 and 1.69 times respectively at 2 h, and that of IL-6 gene 3.7 times compared with the control at 8 h. However, the vesicles inhibited the expression of macrophages IL-10 gene, had no influence on the Frizzled5 receptor expression and could induce CaMKⅡ phosphorylation. Conclusions Exosomes-like vesicles can up-regulat macrophages expression of inflammatory cytokines genes, and increase the secretion of inflammatory cytokines by activating the Wnt5A- Ca2+ signaling pathway.