A Role For NO In The Immunosuppressive Effect Of Human Mesenchymal Stem Cells

Some study showed that mesenchymal stem cells(MSCs) can reduce T lymphocyte proliferation. In peripheral stem cell transpantation for treatment malignant haematological disease,auto MSCs and allogeneic MSCs share the same effect for the patients,whom showed rapidly haematopoietic restoration and a lowest appearance of bleed or infection,escaped from bone marrow restrainment and GVHD. The application of MSCs decreased the complication of stem cell transplantation. There are growing data showing that MSCs have immune regulation function,especially the inhibitory effects on lymphocyte proliferation. However, the mechanisms of the immunosuppression are still obscure. A report demonstrated that STAT5 phosphorylation in mice spleen lymphocyte was suppressed in presence of MSCs and that NO was involved in the suppression of STAT5 phosphorylation and T cell proliferation . In MLR cultures with allogenic lymphocyte as responder cells,NO was involved in the suppression of lymphocyte proligeration in the presence of hMSCs or not . Base on these,MSCs were isolated and cultured from healthy donors routinely. MLR cultures were set up with allogenic lymphocyte as stimulators ,To study effects of NO in MSCs on allogeneic T lymphocyte proliferation ,cell apoptosis and the expression level of FOXP3 mRNA in lymphocyte.MSCs were obtained from iliac crest aspirates from healthy donors,MSCs were isolated from bone marrow cells and cultured using a previously reported method of adherence selection. MSCs were recovered from passages 4-5 for experiment. MLRs cultures were set up with peripheral lymphocytes as responder cells and mitomycin C-treated allogeneic lymphocytes as stimulators .In MSCs/MLRs coculture experiments,MSCs and lymphocytes were cocultured as the proportion 1:10 with or without L-NAME. After 4 days,lymphocytes proliferation was determined by CCK-8 assays,NO scretion in coculture supernatant was determined by a Griess reagent kit .Annexin V analysis was used for identification of cell apoptosis .the expression level of FOXP3 mRNA was tested by real-time quantitative PCR(RT-PCR).The experiment showed that:l In MSCs/MLRs coculture experiment,the lymphocytes proliferation decresed apparently and NO production increased obviously compared with control group(P<0.05);In parallel experiments,L-NAME was added,the lymphocytes proliferation restored partially and NO production decreased partially.2 Proportion of apoptotic cell population of MSCs treated MLRs was (20.83±1.69)%,after L-NAME added MLRs the proportion was (9.56±0.96)%,which was decreased obviously compared with the group of MSCs treated MLRs. 3 RT-PCR showed MSCs-treated MLRs had much higher FOXP3 mRNA expression compared with control group( P<0.05),after L-NAME added ,FOXP3 mRNA expression decreased.The conclusions were that NO production in human MSCs was relation to lymphocyte proliferation ,cell apoptosis and FOXP3 mRNA expression .and hMSCs may perform their immunosuppressive ruction by NO production in vitro.