Preliminary study of plasma Exosomes biological characteristics and plasma Exosomes macrophage function
|Course||Clinical Laboratory Science|
|Keywords||Plasma exosomes -like bodies M I macrophages Wnt5a pathway|
Objective: To research in human plasma Exosomes biological characteristics, understanding the plasma Exosomes role in the immune system. 2 obtained by the differentiation of a small body and in vitro induction of the human plasma Exosomes M Ⅰ macrophages were incubated study macrophage Wnt5a pathway is activated plasma Exosomes body. Method: 1. Ultracentrifugation and ultrafiltration the isolated exosomes purified plasma-like bodies, to obtain sterile plasma exosomes-like bodies. Morphology of plasma Exosomes; streaming plasma the Exosomes molecular phenotype observed by transmission electron microscopy; Exosomes the Western blot detection of plasma proteins. 2. PBMCs, cultured in vitro and then type Ⅰ macrophages isolated from the peripheral blood of healthy people. Cell morphology and by flow cytometry cell surface molecule to determine whether the cultured cells into macrophages by microscopic observation. Exosomes in plasma-like small body type Ⅰ macrophages mixed culture extract macrophage RNA was amplified by reverse transcription and electrophoretic relatively unaffected plasma exosomes kind the the small stimulated macrophages stimulated macrophage inflammatory factor gene changes, such as: IL1β, IL6, TNFα, of IL10. The streaming plasma exosomes like bodies to deal with the different time periods of the macrophage intracellular Ca2 changes, which Wnt5a stimulation of the macrophage intracellular Ca2 changes do positive control group. Western Blot method to detect the plasma exosomes macrophage-like small stimulation CamK Ⅱ whether phosphorylation. Results: 1. Ultracentrifugation from the plasma of healthy people to obtain Exosomes. Small body morphology was observed by transmission electron microscopy, these were highly homogeneous, round vesicles; CD63 antibody-coated magnetic beads capture the plasma Exosomes-like bodies of CD81-positive rate of 73.68%, 17.83% of the plasma Exosomes like small expression of CD86, HLA-DR positive rate was 60.63%, plasma Exosomes like bodies also express adhesion-related molecules CD54 and CDllc, positive rate of 14.66% and 17.83%, respectively; the plasma the Exosomes rich have CD63 and CD81 protein, and bodies were also found to contain HLA-DR, CD80, CD86 and Wnt5a molecule experiments. By the flow detection found Exosomes macrophages were incubated for a period of time, raised the the macrophage cytosolic Ca2 average fluorescence intensity. RT-PCR detected Exosomes pro-inflammatory cytokines such as macrophages promote expression of IL1β, IL6, TNFα, peaked at 2h, IL1β, TNFα gene expression regulated expression of 0.85 times and 1.69 times respectively, compared with the control group, IL6 gene upregulated in 4h peaked, is 3.7 times that of the control group, and inhibit the expression of inflammatory cytokine IL10 suppression. RT-PCR detected the macrophage expression Frizzled5 receptor, but of Exosomes its gene expression impact. Western Blot detection P-CamK Ⅱ protein found the Exosomes make the CamK Ⅱ phosphorylation. Conclusions: 1. Bodies in the plasma with the outside on the size and morphology of exosomes like bodies similar. Such a small expression exosomes-like the small body functions molecules such as HLA-DR, CD80 and CD86, and is rich in functional protein plasma exosomes-like bodies may be secreted by a variety of cells, and plays an important role in the immune system. Preliminary study found that plasma exosomes kind of small physical raised macrophage inflammatory factor gene expression, phosphorylation in and CaMK Ⅱ protein in the signaling pathway is, may be by Wnt5a-Ca2 signaling pathway activated macrophages promote inflammatory cytokine secretion.