The Immunoregulatory Effects Of Bone Marrow Mesenchymal Stem Cells On Allogeneic Peripheral B Lymphocyte

Object To study the immunoregulatory effects of bone marrow mesenchymal stem cells(MSCs)on allogeneic peripheral B lymphocytes in vitro.Methods Mononuclear cells were acquired from bone marrow by density gradient centrifugation.Then,they were seeded at 2×10~5个/ml in DMEM-LG supplemented with 10 %fetal bovine serum(FBS)in culture flask.The medium was changed first time after 48 hours,and once every 2 or 3 days thereafter.When the cultures reached nearly 90%of confluence,cells were passaged with routine methods.Flow cytometry was used to detect the cells surface antigens,include CD3,CD4,CD8,CD13,CD22,CD33,CD34,CD44,HLA-DR.Mononuclear cells were isolated routinely from peripheral blood,then monocytes were eliminated by L-leucine methy ester;remained T lymphocytes were eliminated by AET-SRBC rosette method.The action of MSCs and its supematant on B lymphocytes proliferation in the presence of antihuman immunoglobubin M goat antibodies(Anti-IgM) was investigated by MTT;the IgG,IgM in the supernatant were detected by ELISA.The percent of apoptosis B lymphocytes,co-cultured with MSCs for 24 hours or 48 hours,was assayed by FACS.Results In our study,it was observed that mononuclear cells from bone marrow have adhered to culture flask after 2 to 3 hours culture period.The quantity of adhered cells no further increased after 24 hours.After about a 3-week primary expansion period,MSCs nearly reached confluence,and these cells had a fibroblast-like morphology.After passaged, those adhered cells were homogenous population and proliferated rapidly.Analyzed by flow cytometry,MSCs were demonstrated that they were uniformly positive for CD13,CD44,but negative for CD3,CD4,CD8,CD22,CD33,CD34 and HLA-DR.The result demonstrated that there were no hematopoietic cells in these cells isolated and cultured in vitro.The study shows when Allo-MSCs were added to B lymphocytes stimulated by Anti-IgM in vitro,MSCs and its supernatant inhibited B lymphocytes proliferation and Ig secretion.The inhibitory effect depended on the amount of MSCs and condition of its supernatant.The A value orB lymphocytes co-cultured with MSCs at different ratios,group MSCsⅡ(B:MSCs 10:1)and group MSCsⅢ(B:MSCs 1:1)were both significantly lower than that in control group(both P＜0.01),and group MSCsⅢsignificantly lower than that of group MSCsⅠ(B:MSCs 50:1)(P＜0.05).B lymphocytes were co-cultured with different condition of MSCs supernatant after 3 days,group 50%supematant was significantly lower than that in control group(P＜0.05),and significantly lower than group 12.5%(P＜0.05).Compared with the level of Ig of culture supematant of control group,the level of IgM of group B:MSCs 1:1 and group 50%super were decreased(P＜0.01),only the IgG of group B:MSCs 1:1 was significantly lower than that in control group(P＜0.05).MSCs didn't induce apoptosis of B lymphocytes.The apoptosis rates ofB lymphocytes co-cultured with MSCs for 24h or 48h,in presence of Anti-IgM or not,were not a significant difference among the four groups.The effect of MSCs suppressed on B lymphocytes in vitro was reversible.B lymphocytes and MSCs co-cultured for 3 days,then B lymphocytes were isolated from MSCs and re-stimulated by Anti-IgM.Compared with group pure B lymphocytes,the value of A was significant different:(P＜0.01).Conclusions MSCs have immunoregulatory effects on B lymphocytes,and its mechanisms were complex,not only correlating with the concentration of MSCs but also the action between cells and the secretory cytokine of MSCs.