ABSTRACT
Objective To isolate and identify exosomes from serum samples of the patients with polymyositis / dermatomyositis (PM/ DM),and analyze their protein composition preliminarily.Methods Exosomes from serum samples of the patients with PM/DM were isolated and purified by the ExoQuickTM kit.The morphological characteristics and particle size of exosomes were determined by transmission electron microscope (TEM) and NanoSight analyzer,respectively.The surface markers of exosomes such as CD9,CD81 and Flotillin-2 were identified by western blot.The concentration and composition of exosome protein were determined by the BCA method and SDS-PAGE,respectively.Results The exosomes from serum samples of PM/DM patients displayed round or oval vesicles with membrane structure under TEM,and their diameter range was about (92 ± 67) nm.western blot showed that these exosomes expressed CD9,CD81 and Flotillin-2.The total protein concentrations of exosomes in the patients with PM/DM and healthy controls were 14.68 (6.00,32.55) μg/μL and 14.09 (8.00,23.28) μg/μL,respectively.SDS-PAGE showed that high-abundance proteins enriched in 55-70 kD in both PM/DM patients and healthy controls,and that there were different bands in 40-55 kD between them.Conclusion Exosomes are isolated from serum samples of the patients with PM/DM successfully,and their protein concentration and composition are analyzed preliminarily,which provides the experimental evidences for further finding differential proteins.
ABSTRACT
Objective To investigate the effect of miR-146a on the proliferation and interleukin (IL)-2 production of T helper cells from primary biliary cirrhosis (PBC) patients.Methods MiR-146a in the peripheral blood mononuclear cells (PBMC),monocytes,T helper cells,cytotoxic T cells and B cells from 20 confirmede PBC patients and age/sex matched healthy controls were detected by quantitative PCR.By gainand-loss of function,the miR-146a's effect on anti-CD3/anti-CD28 activated T helper's proliferation and IL-2 production ability were measured by CCK-8 approach and enzyme linked immunosorbent assays (ELISA),respectively.Statistical analysis were carried out by t-test.Results PBMCs (0.46±0.20 vs 1.00±0.26; t=7.47,P＜0.01),T helpers (0.33±0.13 vs 1.00±0.14; t=6.15,P＜0.01) and monocytes (0.56±0.11 vs 1.00±0.11; t=4.97,P＜0.05),but not B cells (0.91±0.06 vs 1.00±0.14; t=0.97,P＞0.05) and cytotoxic T cells (0.98±0.15vs 1.00±0.12; t=0.22; P＞0.05) from PBC patients had lower miR-146a expression level than that of healthy controls.Inducible up expression of miR-146a was observed in PBC patients'T helpers stimulated with antiCD3/anti-CD28 (1.00±0.18 vs 9.12±2.05; t=8.81; P＜0.01).The activated T helpers from PBC patients had higher proliferative ability [PBC:0.35±0.06 (A); healthy controls:0.26±0.04 (A); t=2.83; P＜0.05] and increased IL-2 production [PBC: (685.60±109.19 pg/ml)]; Healthy controls: [(512.20±72.26) pg/ml; t=2.96; P＜0.05 ] than those of healthy controls.For activated T helpers,the proliferation ability,as well as IL-2 production,was enhanced by miR-146a.Conclusion MiR-146a can down regulate the proliferation and IL-2 releasing of activated T helpers.The reduced miR-146a expression enhances IL-2 production and promotes proliferation of T helper of PBC patients,thus,may be involved in the pathogenesis of PBC.