Plasma Exosomes Biological Characteristics And Plasma Exosomes Macrophage Function In The Impact Study

Objective:1. Study the characterization of plasma Exosomes to understand the functions of Exosomes in the immune system.2. Human plasma Exosomes incubated with MⅠmacrophages induced in vitro to study whether Wnt5a pathway is activated by plasma Exosomes.Methods:1. Plasma Exosomes were isolated from human plasma by ultracentrifugation and then purified by ultrafiltration. Transmission electron microscopy was used to observe Exosomes'morphologies and sizes. FACS was used to analysis phenotypic molecules of Exosomes from plasma. Western Blot was used to detect plasma Exosomes'immunity-related proteins.2. PBMCs were isolated from healthy human peripheral blood and then cultured in vitro to obtainⅠtype macrophages. Cells morphology was observed by microscopy and macrophages'molecules were determined by flow cytometry. TypeⅠmacrophages were cocultured with the plasma Exosomes, then the macrophages'RNA were extracted. Stimulated macrophages'genes were compared with unstimulated macrophages by the ways of RT-PCR and electrophoresis, such as IL1β, IL6, TNFa and IL10. Macrophages were treated with plasma Exosomes for different time, then changes in intracellular Ca2 + were tested by flow cytometry. Changes in intracellular Ca2 + of Wnt5a stimulated macrophages were the positive control group. Western Blot was used to detect whether CamKⅡof macrophages stimulated by plasma Exosomes had been phosphorylated.Results:1. Purifying Exosomes from human plasma involved a series of centrifugations to remove dead cells and large debris, followed by a final high-speed ultracentrifugation. Transmission electron microscopy was used to observe Exosomes'morphologies and sizes. Plasma Exosomes were of typical round shape and homogenous in size. The results of FACS were that the percentage of plasma Exosomes immunoisolated by anti-CD63-coated beads that were positive for CD81 tetraspanin molecules was 73.68%. These Exosomes contained moderate levels of the adhesion molecules CD54 and CD 11c. As for other molecules,17.83% of plasma Exosomes expressed CD86, and 60.63% of plasma Exosomes expressed HLA-DR. The Western Blot showed that vesicles from human plasma were rich in CD63 and CD81. Moreover, the vesicles were found to contain Wnt5a HLA-DR, CD80 and CD86.2. After cocultured with plasma Exosomes, MFI of cellular Ca2+ of Macrophages increased. We found that plasma Exosomes enhanced Macrophages to express genes of the cytokines such as IL1β, IL6 and TNFa, the improved expression of genes of IL1βand TNFa were maximal by 0.85 times and 1.69 times larger than the control at 2h, and the expression of gene of IL6 was 3.7 times compared with the control at 8h. However, plasma Exosomes inhibited macrophages to express genes of anti-inflammatory cytokine IL10. Macrophages also expressed Frizzled5 receptor which wasn't influenced by plasma Exosomes. We also found that Exosomes could make CamKⅡphosphorylated.Conclusions:1. The sizes and morphologies of vesicles from plasma are similar with Exosomes. The vesicles from plasma express immunity-associated molecules, such as HLA-DR, CD80, CD86 and so on. Therefore, plasma Exosomes may be derived from many kinds of cells and play important roles in immune system.2. Plasma Exosomes can promote macrophages to express genes of inflammatory cytokines, make CamKⅡphosphorylated, and increase the secretion of inflammatory cytokines of Macrophages by activating the Wnt5a-Ca2+ signaling pathway.