The effect of CsA on theimmunosuppression of bone marrow-derived mesenchymal stem cells induced by IFN- γ and TNF- α

Abstract

：Objective To investigatethe effects of cyclosporine A on the immunosuppression of bone marrow-derived mesenchymalstem cells (MSCs) induced by interferon-γ(IFN-γ) andtumor necrosis factor-α (TNF-α).MethodsMSCs were isolated, cultured, purified and proliferated from bone-marrow of Balb/cmice.After pretreated with the inflammatory cytokines IFN-γ, TNF-α and IFN-γ+ TNF-α (final concentration of 20 ng/ml), MSCs were co-cultured withspleen cells which were activated by concanavalin A for 72 h.Following groups wereassigned: group 1, spleen cells alone; group 2, MSCs + spleen cells; group 3, MSCs(pretreated with IFN-γ) + spleen cells; group 4, MSCs (pretreated with TNF-α) +spleen cells; group 5, MSCs (pretreated with IFN-γ and TNF-α) + spleen ceils; group 6, CsA + spleen cells; group7, MSCs (pretreated with IFN-γ and TNF-α) +CsA + spleen cells; group 8, MSCs (pretreated with IFN-γ and TNF-α) + 1400 W+ spleen cells.The ratio of MSCs andspleen cells was 1 : 10 with the amount of spleen cells being 5000 and the concentrationof CsA 10μg/ml.The proliferation of spleen cells was accessed with MTT assayand the apoptosis was detected by flow cytometry after 72 h.Real-time PCR was used todetect the expression of iNOS mRNA in MSCs treated by inflammatory eytokines to observeinfluence of CsA on the expression of iNOS mRNA.Results In MSCs pretreated with or withoutIFN-γ, TNF had no significant inhibitory effects on the proliferation ofspleen cells (P>0.05).However, IFN-γin combinationwith TNF-α, and CsA alone could both inhibit the proliferation of spleencells, but inhibitory effect of the former could be attenuated by the latter one(P<0.05).The apoptosis rate in groups 5, 6, and 7 was higher than in groups 1, 2, 3,and 4 (P<0.05), and that in group 5, and 6 significantly higher than in group 7(P<0.05).MSCs pretreated with combination of IFN-γ and TNF-α expressed high level of iNOSthan in control groups, which could be suppressed by CsA (P<0.05).The proliferation ofspleen cells in group 8 was also inhibited.Conclusion CsA inhibits the immunosuppressionof MSCs induced by combination of IFN-γand TNF-a viarepressing the expression of iNOS.