| Dendritic Cells (DCs) are important antigen-presenting cells (APC) of the immune system, and are the most potent professional APC known by far. Some of their functions include initiating immune response, stimulating T, B activation and also they play a crucial role in anti-tumour, anti-inflammation, implantation rejection and autoimmune diseases. Furthermore, recently studies show that DCs can modulate immune response and induce immune tolerance. Objective: To establish a stable culture system for the differentiation and maturation of DCs from peripheral blood monocyte cell in vitro, to investigate their phenotypic expression using Flow cytometry and functional analysis by MTT assay. Present study will provide a reliable method to obtain functionally active DCs and found a basis for the further studies of their role in the immune tolerance as well as their anti-tumour effect. Methods: The monocytes were isolated from human peripheral blood monouclear cells(PBMCs)through attachment to the plastic surface. The acquired monocytes were subjected to GM-CSF and IL-4 for 5-7 days culture to induce DCs differentiation. The differentiated DCs were then exposed to GM-CSF and TNF-αfor another 5-7 days to mature. The morphology of DC generated in dishes or cell factories was evaluated under an inverted phase contrast microscope and photographically documented. Flow cytometry was used for phenotypic analysis of CD14, CD83, CD1a, CD80, CD86, HLA-DR and CD25. The phagocytic function was analyzed through the uptake of neutral red by immature DC. Secondary mixed lymphocyte culture was carried out by using immature DC-conditioned T cells as responders and T cells from the same donor of DC as stimulators. We also analyzed the stimulatory function of allogeneic DC upon T cells and their anti-tumor growth effect. Results: Loosed attached cells appeared after 48 hours induction using GM-CSF and IL-4, but no obvious projections were seen. The cells became... |
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