The Experimental Study Of Antitumor Immune Response Against Intracranial Gliomas Induced By Dendritic Cells Pulsed With Human High Immunogenicity Gliomas Cells

Objective: 1. To observe the statement of the major histocompatibility complex class I(MHC class I)molecules expression on the membrane of human in situ gliomas after induced by recombinant human interferon-gamma(IFN-γ) and heating. 2.To investigate whether the human in situ gliomas cells with high immunogenicity could enhanced the capacity of DC from human cord blood mononuclear cells on initiating T cell-dependent specific anti-tumor immunity.Method:The human in situ gliomas was isolated, cultured with enzymatic digestion and identificated used the technique of cytohistoimmunoreactivity. Flow cytometry was applied to analyze the expression rates of MHC class I on the membrane of the human in situ gliomas cultured in differerent time after induced by IFN-γat 500U/ml for 48 hours and then heat shock at 43℃for 2 hours. Cord blood was collected under sterile condition and the cord blood mononuclear cells(CBMCs) were separated by centrifugal in density gradient. CBMCs were cultured with GM-CSF+IL-4 and cellphenotype was analyzed with CD83,CD86 antibody by using immunofluorescence assays. DC from human cord blood mononuclear cells was pulsed with the human in situ gliomas cells and then tested for the capacity of initiating T cell-dependent anti-tumor immune responses using cell counting kit-8 (cck-8) in vitro.Result:1.Twenty primary gliomas were cultured with enzymatic digestion. the tumor cells were purified and identificated by a immunofluorescent study using anti-glial fibrillary acidic protein (GFAP) antibody. 2. The expression rates of MHC class I which maintained at least a week on the membrane of the human in situ gliomas cultured for 2 hours was 91.7% and topmost in a week after induced by IFN-γat 500U/ml for 48 hours and then heat shock at 43℃for 2 hours, which preliminary predicted the feasibility of production in situ gliomas vaccine 3. In the presence of GM-CSF and IL-4, the cells with typical morphological properties of DC at the 7th day were observed. The expression of CD83,CD86 was significantly higher on the 10th day. 4. The DC of experimental groups could obviously initiate T cell-dependent anti-tumor specific immune responses with different ratios compared with control groups(p<0.05) , The killing rate was the highest at the rates of 80:1.Conclusion:1. The method of digestion cultivation can successfully culture primary gliomas. 2. The immunogenicity of the human in situ gliomas cultured for 2 hours was the strongest which MHC class I expression lasted at least a week after induced by IFN-γat 500U/ml for 48 hours and then heat shock at 43℃for 2 hours. 3. Dendritic cells was successfully induced from cord blood mononuclear cells. 4. The DC pulsed with the human in situ gliomas cells could initiate stronger T cell-dependent anti-tumor specific immune responses, which provided the experimental basis for the development of dendritic cells vaccine on this type.