| Bone marrow mesenchymal stem cells (BM-MSCs), as a kind of non-hematopoietic tissue stem cells, are contained in bone marrow and have the capacity of self-renewal, high proliferation and multilineage differentiation. Recent researches have shown that besides supporting the survivor of hematopoietic stem cells in vitro and promoting reconstitution of hematopoietic function in vivo, BM-MSCs could prevent graft-versus-host disease in bone marrow trnasplantation.Hematopoietic stem cell transplantation, especial allogeneic hematopoietic stem cell transplantation is the effective method for treatment of leukemia, aplastic anemia and malignant hematologicaldiseases. But graft reject and graft-versus-host disease remain significant obstacles to a successful outcome in allogeneic hematopoietic stem cell transplantation. Though there are many kinds of prophyraxis and treatment, their effects are not satisfactory. As BM-MSCs are characteristic of easy acquisition, self renewal and high proliferation, they will be a important tool of anti-reject and prevent graft-versus-host disease on condition that the effect of BM-MSCs on graft-versus-host disease is testified.The main mechanism of the effect of BM-MSCs on graft-versus-host disease is that they could suppress proliferation of T-lymphocyte in response to stimuli. Several reports have shown that when autologous or allogeneic BM-MSCs are added to T-lymphocytes stimulated by allogeneic irradiated DCs, allogeneic peripheral lymphcytes or polyclonal activators, a significant and dose-dependent reduction of T-cell proliferation was evident. CD4+ and CD8+ T lymphocytes were equally inhibited by BM-MSCs, and the inhibition is not due to induction of apoptosis of T-lymphocytes and is likely due to the production of soluble factors of TGF- 1 and HGF by BM-MSCs. But some research shows that the inhibition is not due to the production of IL-10, TGF- l and prostaglandin E2 by MSCs. Furthermore, other report show that the suppressive effect of BM-MSCs is not mediated by soluble factors, but requires cell contact. Sothe suppression mechanism on proliferation of T-lymphocyte by BM-MSCs is still to be identified.In this study, we tested the inhibitory effect of BM-MSCs on proliferation of T-lymphocyte stimulated by phytohemagglutinin (PHA), and explored the mechanism(s) underlying BM-MSCs suppression by analysis the levels of TGF- 1 and IL-10 in culture supernatant and evaluating the MSCs-induced T-lymphocytes apoptosis.In this research, a bone marrow aspirate was collected and mononuclear cells were taken by density gradient centrifugation, and plated in human MSC medium consisting of Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG), supplemented with 10% FBS. After allowing 48 hours for adherence to culture flask, nonadherent cells were removed. After a 14-day primary expansion period, MSCs nearly reached confluency, and these cells had a fibroblast-like morphology. Confluent cultures could be processed further by trypsinization and expansion through sequential passages to confluency.MSCs at passage 3 were analysed by flow cytometry, MSC were uniformly positive for CD29, CD44 and CD 166, negative for CD 14, CD34, CD45 and HLA-DR. The result verified that there were not hematopoietic cells in these cells.The study shows that when unrelated donor BM-MSCs were added to T-lymphocyte stimulated by PHA in vitro, a significant (F=65.641, P<0.01) and dose-dependent (r=0.998, P<0.05) reduction of T-cell proliferation was evident. The stronger inhibitory effect was seen if BM-MSCs were added at the beginning of the 5 day culture, and the effct declined if BM-MSCs were added on day 3.The percentage of Annexin V+/VCD3+ of peripheral blood mononuclear cells was detected by flow cytometry. The results showed that after 5 days of cultured with BM-MSCs and PHA, the percentage of Annexin V+/CD3+ of peripheral blood mononuclear cells did not increased compared with that of the control group (P=0.310).There are no differences in the level of TGF- 1...
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