Effect Of The Immunological Properties Of Rat Bone Marrow Mesenchymal Stem Cells By Tumor Necrosis Factor-α In Vitro

Aim: Mesenchymal stem cells are able to differentiate along multiple lineages,they are largely studied for their potential clinical use.This study separated and expanded MSCs in vitro-culture,,investigated whether the immunosuppressive properties of MSCs could be of therapeutic value to inhibit reactive lymphocytes in autoimmune diseases such as rheumatoid arthritis (RA).The effects of MSCs by Tumor Necrosis Factorαwhich secreted by monocyte/macrophages and fibroblasts and plays a central role in RA would be detected.Methods: Separated MSCsfrom bone marrow by density gradient centrifugation ,purified via adhering to the culture plastic, and identified by flow cytometric analysis . The effects of MSCs on the mitogen-induced lymphocytes proliferation and mixed lymphocyte reaction with or without TNF-αare studied through MTT.Secretion changes of IL-6 with or without TNF-αin culture supernatants was determined by ELISA.Results: MSCs could be isolated from bone marrow low-density mononuclear cells expanded in vitro with exhibiting a typical fibroblastlike morphology. Flow cytometry was performed to analyze the expression of molecules of MSCs showed that MSCs expressed CD29,(94.45%),CD105+(87.36%)and did not express CD14,CD34,CD45,CD29.Using a MLR,MSCs were able to suppress the proliferation of allogeneic splenocytes in a dose-dependent manner(1×105,5×104 ,1×104) (P<0.05).Whenever addition of 1×105 MSCs(P<0.05)or MSCs culture supernatants inhibited the ConA-induced proliferative response of lymphocytes(P<0.05).When cultured in the presence of TNF-α,MSCs were unable to suppress the proliferative response of lymphocytes in MLRs, TNF-αalong had no effect on the allogeneic reaction.However,the concentrationsof IL-6 were greatly enchanced in the presence of TNF-αcompared with absence(P<0.05).Conclusion: In this study we effectively isolated and purified by density gradient centrifugation and adhering to the culture plastic and expanded satisfactorily in L-DMEM with 10% bovine serum, which meaning that the methods used to isolate, purify, culture and amplify MSCs are simple, stable and effective; Large numbers of MSCs showed negative immunomodulatory functions and inhibited the mixed lymphocyte response and induction of transformation in vitro;thus the addition of TNF-αwas sufficient to reverse the immunosuppressive effect of MSCs on lymphocytes proliferation,and this observation was associated with a statistically significant increase in interleukin-6 secretion.The therapeutic applications of MSCs for autoimmune diseases needed to be further confirmed.