| BackgroundRheumatoid arthritis (RA) is a mutilation autoimmune disease, which was characterized by synovial hyperplasia, articular cartilage degradation and destruction of bone. T/B Lymphocyte, balance of Th1/Th2 and Cytokines play important effects on pathogenesis of RA. The international acknowledged treatment is the early combination of disease-modifyling anti-rheumatic drugs (DMARDs). However, the clinic remission rate is only 40-70% and the drugs are helpless when joint deformity. Bone marrow mesenchymal stem cells (BMSCs) are non-hemopoietic stem cell in bone marrow. They have ability of self-renewal, high-proliferation and multi-directional differentiation. Investigation supports that they can regulate immunological function, except for helping hemopoiesis and promoting hemopoietic functional reconstruction. So they have general utilization perspective in treatment of autoimmune disease (AID) and induction of transplantation tolerance. It will be a new perspective of RA tresatment.ObjectiveObserve the effect of BMSCs on immunocyte in immune organ of collagen- induced arthritis (CIA), by detecting the proliferation of lymphocyte, expression of Foxp3 mRNA and the level of CD4+CD25+ T cell, thereby explaining the mechanism of immunoloregulation of BMSCs in vivo.Methods1. CIA rats model madding Wistar rats were raised and received mixed liquor of cattleⅡcollagen and Freund's complete adjuvant (CFA).2. The isolation, culture and subculture of BMSCs BMSCs were isolated from the bone marrow of Wistar rats with density gradient centrifugation and screening the cells adherent onto the glass surface, and then cultured in L-DMEM medium supplemented with 10% fetal bovine serum. When cell fusion was to 80% and passaged at a rate of 1 to 3 for expansion. Flow cytometry detects BMSCs'surface antigen CD44, CD71, CD31 and CD45 expression, through the exclusion of the cultured cells for identification of BMSCs.3. Grouping 40 CIA rats were divided to 5 groups randomly. The treatment groups contain the early and advanced stages, which mean that the period of BMSCs transplantation is respectively the first and the second time of receiving mixed liquor of cattleⅡcollagen and Freund's complete adjuvant. Meanwhile, there are normal control and model control. The latter also contains the early and advanced stages, but the difference lies in sodium chloride transplantation.4. BMSCs transplantion All transplantations use the intravenous injection of caudal vein. The cell population of BMSCs is 1×107/kg. The time of executing is 42 days after transplantation.5. Index detection Activate the lymphocyte of CIA rats'spleen with ConA and LPS. Detect the proliferation of lymphocyte with CCK-8. Observe the expression of Foxp3 mRNA by RT-PCR. Flow cytometry is used to assay the level of CD4+CD25+ T cell.Results1. The effect of allogene BMSCs to T lymphocyte The proliferation of spleen T lymphocyte is higher in the model control than in the normal control. And that of the treatment group is lower than the model control. Meanwhile, the proliferation of spleen T lymphocyte is lower in the early treantment group than in the advanced treatment group. BMSCs have effect in inhibiting the abnormal proliferation of T lymphocyte in vivo and the early treantment group is better than the advanced treantment group.2. The effect of allogene BMSCs to B lymphocyte The proliferation of spleen B lymphocyte is higher in the model control than in the normal control. And that of the treatment group is lower than the model control. However, the difference of the proliferation of spleen B lymphocyte between the early and advanced treantment group is not statistically significant. BMSCs have effect in inhibiting the abnormal proliferation of B lymphocyte in vivo.3. The effect of allogene BMSCs to the level of Foxp3 mRNA The expression of Foxp3 mRNA is lower in the model control than in the normal control. And that of the treatment group is higher than the model control. However, the difference between early and advanced treantment group is not statistically significant. BMSCs can promote the expression of Foxp3 mRNA in CIA rats.4. The effect of allogene BMSCs to the level of CD4+CD25+ T cell The level of CD4+CD25+ T cell is lower in the model control than in the normal control. And that of the treatment group is higher than the model control. Meanwhile, the level is higher in the early treantment group than in the advanced treantment group. BMSCs can raise level of CD4+CD25+ T cell in CIA rats and the early treantment group is better than the advanced treantment group.ConclusionsThe mechanism of BMSCs to heal CIA rats maybe involve inhibiting the abnormal proliferation of lymphocyte and promoting the level of Foxp3 mRNA and CD4+CD25+ T cell. And the early treatment is more effectively. This provides the experimental and theoretical basis for treating RA with the immunoregulation of BMSCs.
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