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Study on Immune Mechanism of Human Marrow Mesenchymal Stem Cells Adjusting Dendritic Cells for Treatment of Aplastic Anemia

Author: XiaoYang
Tutor: ZhangLeQin
School: Guangzhou Medical College
Course: Pharmacy
Keywords: Bone marrow mesenchymal stem cells Dendritic cells Aplastic anemia Cell culture
CLC: R556.5
Type: Master's thesis
Year: 2010
Downloads: 61
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Abstract


Background: regeneration obstacles anemia (aplastic ANEMIA, AA, referred to as re-impaired) pathogenesis is more complex in recent years, research think hematopoietic stem cells (hematopoietic stem cell, HSC) intrinsic proliferation or differentiation defects, abnormal hematopoietic micro-circulation system and immune function disorders, alone or combined effects of the three mechanisms in different individuals, leading to hematopoietic failure. Now that AA is a target goal hematopoietic system autoimmune disease in which abnormal T-cell function plays a key role. Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) are a class of self-renewal capacity and immunomodulatory effects of stem cells derived from bone marrow stroma, many clinical studies report that MSC can treat some autoimmune diseases, such as systemic lupus erythematosus, scleroderma, graft-versus-host disease (GVHD). Recent studies showed that MSC regulate the possible mechanisms of the immune response: MSC has a unique immune phenotype, do not express MHC molecules II and FasL expression or very low expression of MHC molecules, costimulatory molecules B7-1, B7-2, CD40 expression CD40L, so it having low immunogenicity, it is difficult by the immune system to recognize. In vitro experiments showed, MSC can inhibit T cells in a mixed lymphocyte culture or mitogenic stimulation of proliferation. Cellular immune response of soluble exogenous antigens can not directly trigger the immune response of T cells, is presented to T cells in a specific manner by antigen-presenting cells (antigen presenting cell, APC), and T-cell activation and production of cytokines associated with different types of APC. Dendritic cells (dendritic cells, DCs) are the most powerful in the body known allied APC, directly stimulate the proliferation of naive T cells (Naive T cells) in vitro and in vivo. MSC proliferation of T cells in patients with AA domestic only see a small amount of reported MSC by adjusting the DCs to influence the proliferation of T cells in patients with AA, domestic have not been reported, and the mechanism worthy of further study. Research purposes: (1) studying whether the MSC on the the AA patient's T cells to inhibit proliferation; (2) to investigate the MSC whether or not the impact of AA patients with T-cell proliferation by regulating the differentiation and maturation of DCs. Research: (1) normal human bone marrow MSC and AA patients with T-cell co-culture, flow cytometry before and after the T cell surface antigens and T cell subsets in co-culture, as well as immune-related cytokines IL- 2, IL-4, IL-10, IFN-gamma in the culture before and after the change in expression (ELISA method), Research MSC AA patients with T-cell activation and proliferation. (2) from normal human peripheral blood mononuclear cells induced by DCs co-cultured immature DCs in the lipopolysaccharide (LPS) stimulation of normal human bone marrow MSC detected before and after flow cytometry DCs co-cultured with or without MSC surface antigen and costimulatory molecules CD80 antigen CD83 expression and mature markers of MSC DCS development. (3) normal human peripheral blood mononuclear cells induced by DCs, mature DCs with normal bone marrow MSC co-cultured for LPS stimulation, the DCs surface antigen and costimulatory molecules CD80 and mature marker antigen CD83 expression (flow cytometry) to study the impact of the MSC mature DCs. Research methods: (1) in vitro culture of normal MSC, cell morphology was observed and detected by flow cytometry its molecular surface antigen CDl05 of, CD29, CD34, CD44, HLA-DR. (2) extract 20 AA patients (without anti-thymus globulin immunotherapy) in peripheral blood mononuclear cells, T lymphocytes nylon wool column separation method to extract the T lymphocytes. Detection and identification of the T cell surface antigens and T cell subsets by flow cytometry. (3) human peripheral blood mononuclear cells were isolated, recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant human interleukin -4 (IL-4) culture conditions prepared DCs. Mature dendritic cells induced by LPS by flow cytometry the DCs mature before and after phenotypic identification. (4) normal human bone marrow MSC co-cultured with aplastic anemia patient-derived T cells, T cell subsets in flow cytometry, as well as immune-related cytokines IL-2, IL-4, IFN-gamma, Expression of IL-10 protein levels before and after coculture (ELISA method). (5) from normal human peripheral blood mononuclear cells induced by DCs, immature DCs in LPS-stimulated and normal bone marrow MSC co-culture, flow cytometry with or without MSC co-culture before and after the DCs surface antigen CD1a, a total of stimulatory molecules CD80 and CD83 expression of mature markers antigen. (6) normal human peripheral blood mononuclear cells induced by DCs, mature DCs with normal bone marrow MSC co-cultured for LPS stimulation, changes in cell morphology was observed under a microscope, flow cytometry the DCs surface antigen CD1a and costimulatory molecules CD80 and mature marker antigen CD83 expression. Results: (1) normal bone marrow mesenchymal stem cells with AA after co-culture of peripheral blood T lymphocytes by flow cytometry T cells CD8 cell ratio decreased from (38.65 ± 1.89)% (29.69 ± 1.62)%, CD4 cell percentage increased from (24.89 ± 1.35)% to (34.89 ± 1.89)%, the difference was statistically significant (P lt; 0.05); ELISA detection of IL-2 protein levels (38.89 ± 4.36)? ng / L dropped to (6.8 Guests 2.12) ng / L; IFN-gamma decreased by (38.46 ± 1.64) ng / L (6.62 ± 1.78) ng / L; IL-4 by (2.8 ± 0.86)? ng / L rise (5.32 ± 1.68) ng / L; IL-10 (2.87 ± 1.12) ng / L rise to (8.28 Shi 1.42) ng / L, the differences were statistically significant (P lt; 0.05) (2) immature DCs after LPS stimulation induced by flow cytometry to detect the normal induction group DCS cell surface expression of CD1a elevated by before induction (2.4 ± 1.2)% to (68.4 ± 12.3)% CD14 expression is reduced by the pre-induction (83.6 ± 1.4)% to (3.5 ± 1.2)%, CD83 (58.7 ± 3.6)% to (72.4 ± 10.6)% (41.2 ± 3.8)%, CD80 raised to (50.6 Guests 6.2)%; co-cultured with MSC group, CD1a expression was still maintaining a high level (82.3 ± 1.6)% (4.4 ± 1.5)%, CD14 expression, CD83 expression (60.8 ± 12.2 )%, CD80 expression (42.2 ± 2.4)%, compared with the LPS-induced no significant difference (P gt; 0.05). (3) the mature DCs MSC co-culture, CD14 (5.8 ± 2.0)% to (62.8 ± 1.6)% of CD1a expression rate (48.6 ± 4.2)% to (30.7 ± 7.8)%, CD83 ( 60.8 ± 12.2)% down to (40.9 ± 6.2)%, CD80 decreased from (50.2 ± 4.8)% to (20.3 Guests 2.6)%, the differences were statistically significant (P lt; 0.05) Conclusion: MSC through the regulation of CD4 and CD8 cells, inhibiting the proliferation of T lymphocytes of patients with aplastic anemia, further experiments show that MSC can inhibit the development of DCS, reversal of mature DCs for immature DCs, suggesting that MSC inhibit the proliferation of T cells in patients with AA possible mechanisms: MSC through the development and maturation of the regulation of DCs reduced the activity of T cells, inhibit their proliferation, has laid a theoretical foundation for the MSC clinical application in the treatment of AA.

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CLC: > Medicine, health > Internal Medicine > Blood and lymphatic system diseases > Blood diseases > Anemia > Aplastic anemia and bone marrow sclerosis,anemia
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