| Objective:Rheumatoid arthritis (RA) is a chronic and systemic inflammatory auto-immune disease characterized by pain, swelling, and inflammation of joints resulting in progressive destruction of cartilage and bone. Though the mechanisms underlying remain to be clarified, cumulative evidence suggests that T cells, B cells and multiple pro-inflamatory cytokines are all involved in the pathogenesis of RA. Thus, that inhibiting antigen-reactive T-cell/B-cell activities and antagonizing pro-inflammatory cytokines are used as critical methods for RA threatment, which also have been proved effective by the successful using biological agents targeting at T cells, B cells and multiple pro-inflamatory cytokines in RA patients. However, these biological agents have not been got satisfying results in the treatment of some patients with refractory RA, and then autologous bone marrow transplantation (Auto-BMT) has been used as an important substitution. Though arthritis in patients with refractory RA has been notably allevated by auto-BMT, the prostecdtive efficacy of auto-BMT is still unknown. Moreover, RA patients-derived stem cells are recently considered to have some functional defects,which raised some suspection on the role of auto-BMT in RA threatment. On the orther hand, cartilage-repairing in the inflammation-infiltrated RA joints is another aspect for RA therapy, which still has not been well-studied so far.The multilineage potential of bone mesenchymal stem cells (BMSCs) and their immunosuppressive nature suggest that allogenic BMSCs may be promising candidates for cartilage repairing and immunotherapy in the treatment of RA. BMSCs-based tissue engineering has obtained satisfactory results in repairing the articular cartilage defects in osteoarthritis (OA) models and patients. In addition, allogenic BMSCs also have been successfully used to treat graft-versus-host diseases (GVHD) in patients who received bone marrow transplantation (BMT) and alleviated a T-cell-mediated auto-immune diseas-experimental autoimmune encephalomyelitis (EAE) in an animal model. However, cell therapy for RA using allogenic BMSCs has not been well-studied.In the present study, we firstly isolated BMSCs from healty adult bone marrow and induced theses cells to differentiate into chondrocyte/cartilage both in vitro and in vivo after expansion in vitro. Then, we explored the therapeutic potentials of BMSCs in RA treatment: the effects of BMSCs on the responses of CII-reactive T cells/ B cells from peripheral blood (PB)/synovial fluid (SF) of RA patients in vitro and on the collagean induced arthritis model by i.v administration were evaluated; we also investigated whether MSCs-differentiated chondrocytes (BMSCs-chondrocytes) possess the same immunological properties as MSCs to explore their cartilage-repairing potentiality in the inflammation-infiltrated RA joints for preventing cartilage re-destruction.Methods (five parts):1. Isolation and identification of BMSCs Bone marrow samples (3~5ml) were obtained from healthy adult human donors, and BMSCs were separated by centrifugation in percoll solution followed by adherence to the plastics cultured in vitro; then, the cell surface antigen were detected by flow cytometry (FCM) and their multilineage potential was confirmed through inducing BMSCs to differentiate into osteoblast and adipocytes; in addition, the morphology, cell growth curve, cell cycle and senescence staining were explored in cells from different passages; furthermore, the microstructure of BMSCs were also observed under transmission electron microscope(TEM).2. Chondrogenic differentiation of BMSCs in vitro and in vivo The micro-cell aggregate was get by centrifugate and was induced by TGF-β1, dexamethasone(Dex) and Vitamin C(Vit C); 21 days later, the micro-cell aggregate were embedded in paraffin, sectioned and stained with HE staining, toluidine blue staining and immunohistochemical staining with anti-type II collagen (CII); the expression of CII in cell plasm were detected by Western blot and The expression of pro CoII mRNA were detected by RT-PCR; in addition, BMSCs-CII scaffod compounds were constructed in vitro followed by cultured in chondrogenesis-inducing medium, and the chondrogenic differentiation of these compounds were observed 8 weeks later after implanting subcutaneously in the athymic mice.3. Influence of BMSCs and BMSCs-chondrocytes on CII-reactive T cells in vitro Paired peripheral blood mononuclear cells and synovial fluids mononuclear cell were isolated from patients with RA; then the effects of both MSCs and MSCs-chondrocytes on proliferation, activation-antigen expression (CD69 and CD25), and cytokines production (IFN-γ, TNF-α, TGF-β1, IL-17A, IL-10 and IL-4) of CII-reactive T cells and T-cell subsets in RA patients were investigated with the stimulation of CII or not by 3H-thymidine-incorporate detection, flow cytometry and ELISA respectively; CD3/Annexin V staining was used to evaluate T-cell apoptosis in the inhibition by FCM; the role of TGF-β1 underlying the inhibition was also investigated.4. Influence of BMSCs and BMSCs-chondrocytes on CII-reactive B cells in vitro Paired peripheral blood mononuclear cells and synovial fluids mononuclear cell were isolated from patients with RA; then the effects of both MSCs and MSCs-chondrocytes on proliferation, anti-CII antibody production,B-cell subsets in RA patients were investigated with the stimulation of CII or not by BrdU-incorporate detection, flow cytometry and ELISA respectively; CD19/Annexin V staining was used to evaluate B-cell apoptosis in the inhibition by FCM; the role of TGF-β1 underlying the inhibition was also investigated.5. Influence of BMSCs by I.V on CIA Rats were isolated and expanded firstly; Collagen-induced arthritis (CIA) model was induced by intradermal injection with chicken CII in complete Freund's adjuvant (CFA) into SD rats,and a second injection of CII in CFA was administered on 8 day after primary immunization; CIA rats received BMSCs by i.v administration were divided into 2 groups according to the time point they received BMSCs after primary immunization: on 7 day and 21 day, respectively; CIA rats who did not receive BMSCs served as positive control; the effects of allogenic BMSCs on the development of arthritis were investigated by a arthritis scoring system and the titre of anti-CII antibody in serum at different time point, together with a histopathological scoring system in ankle joints after rats being sacrificed.Results:1. Isolation, identification of BMSCs and its biological properties The population we isolated showed a fibroblast-like morphology, and expressed multiple cell surface antigens while not expressed cell antigen of hemopoietic stem cell; these cells also retained the capacity to differentiate into osteocytes and adipocytes, which confirmed that the cells we isolated were BMSCs; both morphology and the results of FCM showed that the cells were 90 % and 98 % homogeneous respectively in passage 2 and passage 3; however, BMSCs were found senenscence during sequential passages; in addition, BMSCs were found to differentiate into osteoblast precursors during long-term culture; under TEM,we also found BMSCs were composed of 2 subpopulatons:nave cells and mature cells.2. BMSCs could be successfully induced to differentiate into chondrocytes in vitro and in vivo 21 days later, the HE result showed that the post-inductive cells exhibited a chondrocyte-like morphology. The cells stained with toluidine blue and Col II were positive in the extracelluar matrix. The result of Western blot and RT-PCR showed that the Col II and its pro Col II mRNA were only expressed in post-inductive cells; 8 weeks later after implantation into athymic mice, BMSCs-CII scaffods compounds showed a cartilage-like morphology.3. BMSCs and BMSCs-chondrocytes suppressed CII-reactive T cells in vitro Allogenic BMSCs failed to elicit positive responses of CII-reactive T cells, whereas significantly suppressed CII-stimulated T-cell proliferation and activation-antigen expression in a dose-dependent fashion without inducing T-cell apoptosis; the inhibition was still observed even BMSCs were added as late as 3 days after the initiation of stimulation; moreover, BMSCs inhibited T cells in producing IL-17A, IFN-γand TNF-α(P<0.05), while up-regulated the levels of IL-10 and TGF-β1 (P<0.05) and prevented IL-4 from decreasing(P<0.05); furthermore, Th1 and Th17 were found to be significantly suppressed by BMSCs(P<0.05) while Treg were not significantly upregulated (P>0.05); TGF-β1 was confirmed to play a critical role in inhibition. throughout our study, MSCs-chondrocytes shared the similar properties with MSCs.4. BMSCs and BMSCs-chondrocytes suppressed CII-reactive B cells in vitro Allogenic BMSCs also failed to elicit positive responses of CII-reactive B cells, whereas significantly suppressed CII-stimulated B-cell proliferation and anti-CII antibody prduction without inducing B-cell apoptosis; in addition, BMSCs were also found significantly inhibited the up-regulation of CD27+IgG+non-swiched memory B cells with the stimulation of CII and down-regulation of CD27-IgG+ na?ve B cells(P<0.05); as for CD27+IgD- memory B cells, its elevation in the presence of CII was still suppressed by BMSCs, but insignificantly (P>0.05); TGF-β1 also played a critical role in the inhibition; throughout our study, MSCs-chondrocytes shared the similar properties with MSCs.5. BMSCs alleviated arthritis in CIA BMSCs administration by i.v. fialed to result in the death of CIA rats for graft-versus-host diseases (GVHD), whereas siginificantly allevated arthritis and prevent the development of joint inflammation in the CIA rats(P<0.05); the titre of anti-CII antibody in CIA serum was also significantly suppressed by BMSCs(P<0.05); furthermore, BMSCs were found to significantly prevented the proliferation of synovium and destruction of cartilage in CIA rat(sP<0.05); in addition, the ealier using BMSCs for therapy ,the better it wil be;Conclusion:1. We successfully established the methods in our labaratory for BMSCs isolatation,culture and identification in vitro, and even successffuly induced BMSCs to differentiate into chondrocyte/cartilage both in vitro and in vivo;2. We found allogenic BMSCs and they differentiated chondrocytes could significantly suppressed the responses of CII-reactive T cells and B cells in vitro;3. We found allogenic BMSCs significantly alleviated the development of arthritis and destruction of cartilage in CIA rats;...
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