Effect Of Human Bone Marrow Mesenchymal Stem Cells On Proliferation And Function Of Peripheral Blood Mononuclear Cells And The CD8+T Lymphocytes

OBJECTIVE: To investigate the effect of human bone marrow mesenchymalstem cells (MSCs) on proliferation and function of allogenic peripheral bloodCD8+T lymphocytes. To further explore the inhibitive mechanism of MSCs onCD8+T lymphocytes. To provide experimental basis for clinical application ofMSCs in immune diseases caused by autoimmune diseases and graft versus hostdisease (GVHD).Methods: MSCs were isolated with isopycnic centrifugation and cultured byusing adherent method, and PBMCs were isolated by Ficoll-Pague densitygradient separation. MSCs were identified with morphology, the expression ofsurface markers and capacity of multi-directional differentiation. Theproliferative influence of MSCs on peripheral blood mononuclear cells (PBMCs) stimulated by phytohemagglutinin (PHA) was investigated by using CCK-8in co-culture of PBMCs and MSCs. The effect of MSCs on proliferation ofCD8+T lymphocytes induced by PHA was explored by the combination ofcarboxyl fluorescein acetoacetate succinimide ester (CFSE) assay and flowcytometry. In order to probe the role of cell contact in the inhibition of MSCsand PBMCs, transwell experiments were performed to prevent cell contactwhen PBMCs were co-cultured with MSCs.The CD8+T lymphocytes wereisolated by magnetic cell sorting (MACS). Fluorescence-activated cell sortercombined with CFSE were employed to determine the purity of CD8+Tlymphocytes after the separation. The CD8+T lymphocytes were co-culturedwith MSCs in24-well plates and the RNA was isolated from CD8+Tlymphocytes after72hours of co-culture. Only CD8+T lymphocytes as thenegative controls, and the positive controls were induced by PHA. Expression ofinterleukin-2(IL-2), Granzyme B (Gzmb), Interferon-γ (IFN-γ) mRNA wereevaluated by real-time fluorescence quantitative PCR in CD8+T lymphocytes.Results: MSCs and PBMCs were successfully harvested and cultured in vitro.When PBMCs were co-cultured with MSCs, MSCs could suppress theproliferation of CD8+T cells stimulated by PHA (P<0.05) with MSCs: PBMCsratios of1:5and1:1(P＜0.05). MSCs could not suppress the proliferation ofCD8+T cells stimulated by PHA with MSCs: PBMCs ratios of1:10、1:20and1:50(P＞0.05). Compared to the control with no-Transwell, the group with Transwell that prevents cell contact between MSCs and CD8+T cells whenco-cultured at MSCs: PBMCs ratios of1:5and1:1, there were no difference inthe inhibition of MSCs on proliferation of CD8+T lymphocytes induced by PHA.Magnetic cell sortingwere used to isolate and purify CD8+T lymphocytes. Theexpression of them in CD8+T lymphocytes not stimulated by PHA were low andthey were increased when CD8+T lymphocytes stimulated by PHA. Theexpression of IL-2, Gzmb and IFN-γ mRNA was clearly decreased after treatedwith MSCs and with MSCs: PBMCs ratios of1:5, and the differences werestatistically significant (P<0.05).Conclusions: MSCs can inhibit the proliferation allogenic PBMCs and CD8+Tlymphocytes, the mechanism may be related with cell-cell contact and thesoluble factors.