| Background: Dendritic cells (DCs) are the most potent antigen- presenting cells that belong to in WBC initiated from stem cells, can activate native T cell. But, there's difficulty in sample source and technology for biology therapy using Dendritic cells get from ex-body induce mononuclear cells in peripheral blood. The source of umbilical cord blood mononuclear cell(CBMCs) is rich, but always be rejected. Recently many investigation found that the hematopoietic stem cells in cord blood are more initial and more quantity than in bone marrow and peripheral blood. Some study reported that the mononuclear cells of peripheral blood and cord blood can differentiate into functional Dendritic cells after culture with granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), TNFα. The main reason of immune escape are deficiency of tumor patient's immune funtion, down regulation of processing function for tumor antigen, and low-expression or no expression costimulatory molecules. The immunotherapy using Dendritic cells is one of the main direction for tumor biotherapy. The aims of this study are to examine the method of using GM-CSF, interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO (erythropoietin) for differentiation and increase Dendritic cells in vitro. To explore a new practicable method that Dendritic cells from cord blood mononuclear cell are induced and increased in vitro. To study the effects of dendritic cells from cord blood mononuclear cell pulsed with HL-60 or K562 cells on stimulating the cytotoxic T lymphocyte (CTL) to get specific anti-tumor activity.Objective: 1.The aims of this study are to analyze the composition of umbilical cord blood cells and peripheral blood cells(PBs), and to examine the characteristics of cord blood mononuclear cell from fresh cord blood mononuclear cell and peripheral blood cells 2.To search the method of differentiation and increase dendritic cells in vitro, and to appraise surface antigen from cord blood mononuclear cell. 3.To study the effects of dendritic cells (DC) pulsed with HL-60 or K562 cells on stimulating the cytotoxic T lymphocyte (CTL) to get specific anti-tumor activity.Method: 1. Mononuclear cells of 9 peripheral blood cells cases from healthy adult donors and 12 cord blood mononuclear cell cases were collected. 2.Method of flowcytometry was used to determine the number of dendritic cells and cell surface antigens from peripheral blood cells and cord blood mononuclear cell. The monoclonal antibodies included CD4, CD8, CD19, CD34, CD38, CD83, CD1a, CD11c and CDw123. 3. Cord blood mononuclear cell were cultured for 1,2,3 and 4 weeks with GM-CSF, IL-3, SCF and EPO. 4. The nature of cord blood mononuclear cell before and after culture was freshly determined. Method of flowcytometry was used to determine the number of dendritic cells and cell surface antigens by using monoclonal antibodies, and the ELISA was used to determine the content of IL-12. 5. HL-60 and K562 were treated frozen-thawed, and released its tumor antigen peptides(TAP). dendritic cells pulsed with TAP and coculture to transfer T cell into specific CTL. 3H-TdR release assay was used for evaluate stimulation of dendricic cells. MTT assay was employed to evaluate inhibition rate of CTL on leukaemia cells, respectively.Results: 1. Numbers of CD4~+ cells are 35.36%, CD8~+ cells are 23.44%, CD19~+ cells are 5.07%, CD38~+ cells are 5.07%, CD34~+ cells are 0.15%, CD1a~+ cells are 0.05%, CD11c~+ cells are 13.96%, CD83~+ cells are 3.61%, and CDw123~+ cells are 4.29% in peripheral blood cells. Numbers of CD4~+ cells are 33.43%, CD8~+ cells are 18.9%, CD19~+ cells are 5.98%, CD38~+ cells are 25.04%, CD34~+ cells are 0.59%, CD1a~+ cells are 0.81%, CD11c~+ cells are 18.04%, CD83~+ cells are 5.49%, andCDw123~+ cells are 8.43% in cord blood mononuclear cell. The numerous of CD34~+,CD38,CD1a in fresh cord blood mononuclear cell are more than ones in peripheral blood cells (P<0.01).2.Numbers of CD34~+ progenitors cells are 0.02x105/ml, CD1a~+ cells are 0.01x105/ml, CD11c~+ cells are 4.32x105/ml, CD83~+ cells are 1.31x105/ml, and CDw123~+ cells are 1.41x105/ml in peripheral blood cells. Numbers of CD34~+ progenitors cells are 0.22x105/ml, CD1a~+ cells are 0.27x105/ml, CD11c~+ cells are 5.87x105/ml, CD83~+ cells are 1.94x105/ml, and CDw123~+ cells are 2.73x105/ml in fresh cord blood mononuclear cell. 3. Cord blood mononuclear cell cultured for 1, 2, 3 and 4 weeks with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a~+ CD11c~+ CD83~+ CDw123~+ dendritic cells. Numbers of dendritic cells from cord blood mononuclear cell remarkably generated in 2~4 weeks and then decreased in number. By culture with cytokines dendritic cells increased 10.57~28.24x105/ml in actual numbers. 4. Cord blood mononuclear cell cultured for 1, 2, 3 and 4 weeks with GM-CSF, IL-3, EPO and SCF were shifted along the dendritic cells pathway. Cells showed characteristic morphological features of dendritic cells, including irregular cellular surface, variable size increases, small condensed nuclei, typical cytoplasmic motile processes. As the time went on, number of dendritic cells cultured in all contions increased. 5.The level of IL-2 in fresh cord blood mononuclear cell are lower than those of cord blood mononuclear cell cultured for 1, 2, 3 and 4 weeks with cytokines (P<0.01). The level of IL-2 in cord blood mononuclear cell cultured for 1, 2, 3 and 4 weeks are 6.26±3.09ng/ml,6.92±3.53ng/ml and 5.81±2.87ng/ml.6.The CTL induced by DC pulsed with frozen-thawed lysates were effective to kill HL-60 and K562. Cytotoxicity of CTL to HL60 and K562 were 42.04%±8.46% and 31.25%±11.07%。Conclusion: 1 There are progenitors cells and dendritic cells in cord blood mononuclear cell and presenting cells. Numerous of CD34~+ progenitors cells and dendritic cells in human cord blood mononuclear cell are more than ones in PBs. 2. The cord blood mononuclear cell cultured with cytokines of GM-CSF, SCF, EPO and IL-3 differentiatedinto CD1a+, CD83+, CD11c+ and CDw123+ DCs. 3. The CTL induced by dendritic cells pulsed with frozen-thawed lysates were effective to kill HL-60 and K562. 4. These dendritic cells are very effective as antigen presenting cell and in cancer immunotherapy.
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