Culture And Identification Of Regulatory Dendritic Cells From Mouse Bone Marrow In Vitor

Objective:To induce regulatory dendritic cells from murine bone marrow in vitro and to detect IL-10、SOCS1、TLR4and NF-κB gene expression as well as to explore the mechanism of the immune functions played by theregulatory dendritic cells playing in thus harvested.Methods:1、To isolate and purify of murine bone marrow-derived dendritic cells (DC),which were divided intofour groups,and subsequently cultured in vitro and grouped as A,B,C and D,respectively.2、During the culture,the medium from A and B groups were plused with GM-CSF+IL-4,at the sametime,that from C and D groups with GM-CSF+IL-10+TGF-β1. At day3, the supernatant was abandoned inorder to remove nonadherent cells,meanwhile fresh culture medium and relevant cytokines were added.Half of medium was changed every other day until day8. To promote the differentiation B and D goupswere stimulated with LPS for48h.3、The morphological changes of culture cells were observed under inverted microscope.4、The cells and supernatants from the four groups were collected on day10for flow cytometry, ELISAand RT-PCR.Results:1、In vitro,mouse bone marrow-derive cells were culture with GM-CSF、IL-4、IL-10、TGF-β1and LPS,which can induce a morphological feature of DCs. The dendritic structure of B and D group was moretypical,even the bifurcate of B group was more and thicker.2、Flow cytometry showed that B group expressed higher levels of CD11c((94.181.50)%), MHC-Ⅱ((81.172.45)%), CD80((72.852.24)%) and CD86((24.781.57)%), presenting mature dendritic cells(mDC) characteristics, while A, C and D group expressed lower levels of the above factors, which weredefined as immature dendritic cells (iDCs),regulatory dendritic cells precursor (pDCregs) and regulatorydendritic cells (DCregs).3、ELISA detected of IL-12expression levers.The data of four groups was followed by12.281.67,35.302.36,10.322.23and11.172.82. The result showed that mDC group expressed significantly higherthan the other groups (P<0.05) and no significant difference between the other groups.4、RT-PCR results showed: compared with iDC and mDC, IL-10and SOCS1were higher expression inpDCreg and DCreg (P<0.05),while TLR4and NF-κB were higher expression in mDC and other groups werelower expression (P<0.05).Conclusion:In vitro bone marrow-derived regulatory dendritic cells were successfully induced which have theproperty of immune tolerance. We found that IL-10and SOCS1gene expression played an important role inthe formation of DCreg,opening new avenues for the treatment of autoimmune disease.