血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.

A preliminary study on the regulatory function of human plasma exosomes-like vesicles