| Human postnatal bone marrow contains mesenchymai stem cells (MSCs) capable of differentiating into multiple mesenchrmal tissues such as bone,adipose ,cartilage, and myelosupportive stroma. They are characterized by adherence to plastic, the absence of hematopoietic markers (CD45-/CD34-) and the expression of a specific pattern of molecules (CD166+,CD105+, CD13+, CD29"). They can be isolated from the bone marrow, and can be identified through flow cytometry.Due to the multipotential ability and self-renewal capacity, MSCs may be a promising cell type for tissue engineering, cellular therapy, and somatic genetherapy applications. MSCs are pluripotent cells present in tne bone marrow at low quantity (1 out of 104-105 mononuclear cells). So we must investigate it's biological identities. Immunologically, human MSCs share cell surface markers with thymic epithelium. The presence of these cell surface markers , along with the finding that MSCs are customary residents of the bone marrow micrienviroment, suggest that MSCs may play an important role in the immunoregulation provided by the bone marrow microenvironment. Great potential for MSCs would be derived from the observation that they can exert an immunoregulatory activity. Data from preclinical transplantation studies suggested that MSC infusions not only prevent the occurrence of graft failure, but also prevent the occurrence ofGVHD in HLA-mismatched bone marrow transplantation. They can inhibit T cells proliferation and have an immunodulatory effect, but the effect mechanism has not been defined.In this study, MSCs were isolated and purified from bone marrow by gradient centrifugation on Percoll(density 1. 073g/ml) ,and expanded ex vivo. The purity of MSCs was analyzed by flow cytometry. MSC had strong self-renewal capacity. After primary culture approximately 1-2 X 106 MSCs were obtained from 5X106 MNC of bone marrow. After passage 5, passage 10, we could get 108 and 1010 MSCs. After passage 3, the purity of MSCs was above 98% according to FACS analysis. Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased,but they maintained good puripotantiality. In this work, The stromal feeder layers of irradiated MSCs in different quantity (group A 8X104 MSCs/well, group B 4X 104 MSCs/well, group C 2 X 104 MSCs/well) were cocultured with allogeneic T lymphocytes isolated from peripheral blood. Peripheral blood T cells non-cocultured with MSC acted as control group. CD3, CD4, CD8, and CD25 expressed on T cells were analysed by flow cytometry after cocultured with MSCs at 0 hour,24 hour,72 hour and 7 days respectively.The results showed that in group A and group B CD4+CD25+ T cells and CD8+ T cells of allogeneic T lymphocytes cocultured with bone marrow MSCs increased obviously compared with control group , but between group C and control group there was no difference, and between group A and group B there were no difference. CD3,CD4, CD8 and CD25 expressed on T cells have no difference between experiment group(group A,group B, group C) and control group.The result suggested that CD4+CD25+ regulatory T cells (Treg) and CD8+ T cells were increased apparently when T cells cocultured with more MSCs, and the phenotype of T lymphocyte had no changed when cocultured with less MSCs.The result indicated that MSCs may be can induce immunologic tolerance through phenotype of T lymphocyte changed . MSCs pretreatment might beuseful in the prevention of GVHD and HVGR in allogeneic bone marrow transplantation and provide a new strategy to induce transplantation immunological tolerance.
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