The Research Of Immunological Mechanisms In The Pathogenesis Of Aplastic Anemia

Aplastic Anemia (AA) is a heterogeneous disease characterized by failure of bone marrow hematopoiesis resulting in varying degrees of pancytopenia with a markedly hypocellular bone marrow. Clinically, patients with AA may present with anemia, bleeding and serious infection. Despeite the exacts cuases of AA are unknown, the disease may result from the accumulated effects of multiple noxious exposures of plurpotential stem cells. Potential mechanisms responsible for acquired AA include induced defects in hematopoietic stem cells, failure of the bone marrow microenvironment which impaired production or release of hematopoietic growth factors, and cellular or humoral immune suppression of the marrow. The introduction of immunosuppression therapies such as antithymocyte globulin, antilymphocyte globulin, and cyclosporin A in the treatment of AA has improved the prognosis. Some of 50-80% patients recovered following immunosuppression suggestion that aplasia may be the an immune-mediated effect. T lymphocyte mediated suppression has been considered the most important in the development of AA. Further study T cells, especially abnormal activated T cells, were not only a useful marker to understand the pathegenesis of AA, but my have great implication on clinical application.The activation of naive T cells is the key link of immune response and this process requires the generation of two signals. It postulates that T cells activation requies a first step involving presentation by professional antigen presenting cells ( APCs ) such as dendritic cells and a second step in which Ag-specific, the APCs take up the antigen, process it, and subsequently present the peptides of the antigen on their class II MHC molecules. Inducible co-stimulatory melecules are expressed on those cells when effector T cells recognize the these MHC-bound peptides, and such induced cells can now activate T cells specific for another MHC-bound peptide derived from the same antigen. The inactivation or immunologictolerance occurs when the T lymphocyte interacts with antigen alone. Dendritic Cells(DCs), the most potent professional antigen presenting cells in vivo as yet, are capable of initiating autoimmune or allergic reaction and generate tumor-specific immunity by stimulating the proliferation of naive T lymphocytes. Recent researches suggested that rearrangement of T cell receptor, dendritic cells and abnomal expression of co-stimulatory molecules participated in the development of many autoimmune diseases. Whether there is abnomal expression of TCR V 0 genes, moreover whether DCs initiate AA and co-stimulatory molecules which associate with activation of CD8+ CTL and what the relationship between them are unknown. So it is worthy of researching deeply though there is few study in this field.For all these reasons, we have paid much attention to the expression of TCR V 0 generepertoire in AA Patients, the interaction of DCs with CD34+ cells and the relationship of co-stimulatory molecules among the abnormal CD8 T cell activation lymphocytes. So we cananalyse the reason of abnomal CD8+T cell activation and it's regulating mechanism in AA patients. To investigate the impacts of TCR V ï¿¡ gene repertoire rearrangement , DCs and co-stimulatory molecules on CD8 T activation, proliferation, cytokines production andapoptosis not only help us to understand the immunological factors in the pathogenesis and development of AA, but also have great implication on diagnosis, treatment and prognosis of the disease in particular.To investigate the possible mechanisms in pathological immune response in patients with AA, We use several biologic and immunologic mothods to research the expression of TCR VP gene repertoire of AA patients , phenotype and proliferation of CD34 cell-reactive T-cellclones stimulated by DCs and the possible roles of co-stimulatory molecules mediated abnormal T cells activation. Using specific monoclone antibodys and drugs which target specific TCRV P gene,DCs or co-stimulatory molecule such as LIGHT may be a novel way to treat AA patients.1. Oligoclonal expansion of T cells in periphere blood of patients with AATCR V P subfamily genes in peripheral blood mononuclear cells from 2 cases of normal and 10 cases of AA were amplified by using RQ-PCR.As a control,we also researched SLE,MS,ITP and GD patients by the same method.The results showed that only 2-9 V P genes were found in samples from these patients of AA, and there were different distribution in different patients. TCR repertoire complexity was abnormal in all patients.Samples from 10 patients showed V3 4, 22 , 2, 5, 8, 14 and 24 etc overexpression in these cases of AA patients.Parts of the genes were expansion and part of them were surpressed . These results indicated that clonal expansion of T cells could be found in AA cases and might be related with the pathogenic initiation of AA.2. The role of DC in the mechanism of abnormal T cells activation in AADendritic cells (DCs) were obtained from mononuclear cells of peripheral blood and CD34+cells were isolated from umbilical cord blood microscope. The phenotype of DCs were analysed by Flow Cytometry. we applied DC to uptake antigen of CD34+cells treated by Y -ray irradiation and antigens of apopotosis tumor cells such as XG1 and RAJI . After the stimulation of sCD40L , DCs' ability to activate the proliferation of T cells was tested and the changes of TCR V P gene repertoire of these T lymphcytes were also compared. Moreover the inhibition of different Arsenic trioxide on Dendritic cells and the impact on DCs' ability to activate the proliferation of T cellswere determined by using MTT assay. After uptake antigens of CD34 cells apopotosistumor cells ,DC enhanced it's ability to activate the proliferation of T cells and made these T cells product the restricted usage of TCR V P genes. Moreover we demonstrated that Arsenic trioxide could inhibit DC in dose- dependent manner and down-regulate DCs ability to activate the proliferation of T cells significantly.So we consider that DCs play an important role in pathogenic mechanism in AA and umbilical cord blood transplantation and Arsenic trioxide has the possibility to treat AA.3. LIGHT is highly expressed in BM and PB T cells from AA patientswe used flow cytometry to analyse the expression of LIGHT of lymphocytes in AA bone marrow.LIGHT mRNA in PB T cells from patients with AA was detected by RT-PCR. The results show that the expression of LIGHT in BM and PB T cells in AA especially in SAA patients were increased significantly. So we think that LIGHT plays an important role in the pathogenesis of AA and associates with the abnormal activation andoligo-clonal expansion of CD8 T cells in AA. The research aimed at LIGHT may build a novel way to diagnose and treat AA patients.