ABSTRACT
Objective: To obtain and identify the exosomes derived from human stem cells from the apical papilla (hSCAPs). Methods: hSCAPs were cultured by modified tissue adherence method and the phenotypes were analyzed with stem cell surface markers CD105, CD45, CD44, CD31, CD34 and CD29. The capability of multi-differentiation in hSCAPs was identified by osteogenic and adipogenic differentiation in vitro. Exosomes were isolated from hSCAPs culture supernatants using gradient centrifugation methods. The size of vesicle was assessed by nanoparticle size analyzer. The morphology of exosomes was observed by transmission electronic microscope (TEM), and the expression of exosome molecular markers CD81, CD9, CD63 and TSG101 was analyzed by Western blotting. Results: hSCAPs were positive for the mesenchyme stem cell markers, including CD105, CD44 and CD29 and negative for the hematopoietic markers CD45, CD31 and CD34. hSCAPs could differentiate into osteoblasts and adipocytes. hSCAPs secreted microvesicles which exhibited round vesicle structure with an intact membrane observed by the TEM. The results of nanoparticle size analyzer measurement showed that the diameters of vesicles were ranged from 30 to 100 nm, which were consistent with the results by TEM. Microvesicles could express the molecular markers for exosomes, i.e. CD81, CD9, CD63 and TSG101. Conclusion: The microvesicles were successfully isolated from hSCAPs and identified as exosomes.
ABSTRACT
Objective·To investigate the effect of 3,3'-diindolylmethane (DIM) on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLCs) induced by lipopolysaccharide (LPS) and to study the related mechanism.Methyls· hPDLCs were isolated and cultured,and CCK-8 method was used to detect the effect of DIM on the proliferation of hPDLCs.hPDLCs were randomly divided into 4 groups:blank group (without LPS and DIM),LPS group (10 μg/mL LPS),10 μg/mL LPS+6.25 μg/mL DIM,10 μg/mL LPS+12.50 μg/mL DIM.The cells of all groups were cultured for 12 h.The protein levels of TNF-α,IL-1β and IL-6 in supernatant were detected by enzyme linked immunosorbent assay.The change of mitogenactivated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways were detected by Western blotting.Results· The cell viability was not affected when the DIM concentration was less than 50 μmol/L (P＞0.05).DIM at 6.25 and 12.50 μg/mL reduced the LPS-induced expression of TNF-α,IL-1β and IL-6 at protein levels (P＜0.05).DIM inhibited the activation of the NF-κB signaling pathway.Conclusion· DIM can reduce the LPS-induced inflammatory cytokine expression in hPDLCs via restraining the activation of the NF-κB signaling pathway.