| Adoptive immunotherapy, i.e. the method for inducing anti-tumor immune effector cells, is an important immune strategy to treat malignant melanoma. Studies confirmed that malignant melanoma showed a certain reactivity to LAK, TEL ( tumor- infiltrating lymphocyte ) ,CD3AK ( anti-CD3 McAb-activated killers) ,PAK (PHA-activated killer) ,andNK cells. However, the total effect is not so satisfactory as the activated effector cells only belong to one species and their curative effects are instantaneous. The most recent studies showed that the T-cells derived from the tumor-vaccine-stimulated tumor-draining lymph node (TDLN) have a stronger anti-tumor action after stimulation in vitro with CD3+ CD28+EL-2 and returning to the body . A research group from University of Michigan obtained a significantly curative effect by using a scheme of in vitro stimulation with TDLN cells combined with CD3+CD28+IL-2 to treat the late-phase melanoma and kidney cancer in both animal and clinical experiment.Superantigen SEA is a group of proteins coded by bacteria or viruses, and combined as a whole molecule directly with the outside of the antigen-binding groove of MHC II molecule and the TCRV region of T cell surface antigen-recognition receptor respectively, so as to activate T-lymphocytes a few thousands to tens of thousands times more than common antigens do, without need of APC. Therefore, superantigen SEA is a powerful activator of T cell. Dendritic cell ( DC ) ,as the most important antigen-presenting cell in vivo, can effectively present antigen to and activate T lymphocyte with a high efficiency . As cell surface highly expresses a variety of immune molecules required for T cell activation, DC as the most important antigen-present cell can effectively present and activate T cell with a high efficiency. In view of the unique action of SEA and DC on the activated T cell and tumor immunity, we made use of SEA-activated TDLN-T lymphocytes combined with B16 cell lysate-impacted DC cells to stimulate in vitro and in combination, with the aim of searching a scheme with more optimized specific inducing and killing of the malignant melanoma with high efficiency and explore more effective immunotherapy for melanoma.We constructed a eukaryotic expression vector pcDNA3.1-SEA and transfected it into the B16 cell by means of liposome-mediated transfection method, and then the resultant positive clone was multiplied in culture . The transfection was confirmed to be successful by identification with RT-PCR. BALE /c mice were divided into experimental and control group, which was injected into the tail vein, respectively with the transfected and non-transfected B16 cells, and examined for the number of metastatic tumor focus after two weeks; the results demonstrated that the number of the metastatic tumor focus in the SEA experimental group was notably decreased and different most significantly from the control group (p<0.01) , confirming that SEA can raise the immunogenicity of B16 cells and excite effectively the killing activity of T cell against B16 cell and the immune response to the malignant melanoma.In this study, a high dose of cyclophosphamide ( CY ) ( 200mg/kg of weight) was given to mice by the injection into the tail vein, 8 days after which the mouse splenomegaly was obvious, and the splenocytes were cultured with GM-CSF and IL-4. On day 5 of culture, TNF-a was supplemented to the culture,also 8 days after which a large number of lymphatic DC, (CYSPDC ) were obtained and an average of 7.5+107 DC/mouse also achieved. The cultured cells were observed by phase-contrast microscopy, scanning electron microscopy ( SEM ) and transmission electron microscopy ( TEM ) and all possessed the typical characteristics of DC morphology. Meanwhile, the phenotype of the cultured cells was analyzed by means of FACS and the results showed that the main immunological marker molecules of the mature DCs, such as N418 (CD11c), MHC II; MHC I (H-2Db), B7-2 (CD86) and soon, varied with the time and displayed an ascending tendency...
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