The Manufacture Of Human Glioblastoma Multiforme U251 Cell Vaccine With High Immunogenicity And The Probe Of The Mechanism Of Its Antitumor In Vitro

Objective: 1.To investigate the ability of recombinant human interferon-gamma(IFN-γ) to upregulate major histocompatibility complex class I(MHC class I)molecules expression on the membrane of human glioblastoma multiforme(GBM)U251 cells in vitro. 2.To investigate the ability of β-elemene and/or heat to upregulatethe membrane molecule heat shock protein 70 (HSP 70)expression of GBM U251cells in vitro. 3.To investigate the ability of recombinant human interferon-gammaand/or heat to upregulate the membrane molecule MHC class I and HSP 70expression of GBM U251 cells in vitro. 4.To investigate the manufacture of humanGBM U251 cell vaccine with high immunogenicity and the mechanism of itsantitumor in vitro rudimentally.Methods: Flow cytometry was applied to analyze the expression rates of MHC class Iand HSP 70 on the membrane of GBM U251cells which had been treated withdifferent intervention agents. U251 cells were treated by optimum inducementschemes namely membrane-enriched MHC class I molecule,membrane-enriched HSP70 molecule and the membrane-enriched two molecules mentioned aboverespectively,lastly the mitomycin C-inactivated the treated U251 cells were the threedifferent GBM U251cell vaccines. PBMCs from healthy donator were coincubatedrespectively with the three GBM U251 cell vaccines, then collected as the threedifferent effector cells. The competence of specific killing of the three effector cellswas determined by MTT assay; At the same time, the interdiction and theintercrossing interdiction tests that anti-human HLA-A,B,C and anti-HSP70monoclonal antibody were applied in the specific killing test were carried out;Flowcytometry was applied to analyzed the changing of the proportions of CD4+ andCD8+T lymphocytes; the secretion of IFN-γand IL-2 of the effector cells which hadassaulted the target cells was evaluated by ELISA assay. The ultrastructure of theassaulted target cells was observed by Electron microscope.Results: 1.The expression rate of MHC class I molecules on the membrane ofU251cells was 89.9% and topmost among the experimented groups when U251 cellswas treated by IFN-γat 500U/ml for 48 hours in vitro. 2.The expression rate ofmembrane molecule heat shock protein 70 of U251 cells treated with singleingredients namely treatments with β-elemene at 0.1mg/ml for 2 hours and heat shockat 43℃for 2 hours,41.63 % and 42.18% respectively, were highest among theexperimented groups in vitro. 3.The expression rates of membrane molecule MHCclass I and HSP 70 of the U251cells treated by double ingredients namely IFN-γat500U/ml for 48 hours and then heat shock at 43℃for 2 hours were 96.00% and53.61% respectively, were highest among the experimented groups in vitro. 4.CTLsstimulated by GBM U251 cell vaccine which were the membrane-enriched twomolecules namely MHC class I and HSP 70 molecule had stronger competencespecifically killing wild type U251 cells than those stimulated by themembrane-enriched MHC class I molecule U251 cell vaccine and themembrane-enriched HSP 70 molecule U251 cell vaccine and untreated controlgroup(p<0.05), the proportion of CD4+ and CD8+T lymphocyte of PBMCscoincubated with it had increased dramatically(p<0.05), and could secrete a greatestdeal of IFN-γand IL-2(p<0.05);there existed apoptotic necrosis and oncotic necrosisto a distinct extent in the target cells assaulted by the three different effector cells.Apoptotic cells were observed more in the CTLs group stimulated by themembrane-enriched MHC class I molecule U251 cell vaccine and that sitmulated bythe membrane-enriched HSP 70 molecule U251 cell vaccine than CTLs groupstimulated by the membrane-enriched two molecules namely MHC class I and HSP70 molecule U251 cell vaccine, the oncotic phenomenon were more in CTLs groupstimulated by the membrane-enriched two molecules namely MHC class I and HSP70 molecule U251 cell vaccine than the other two groups.Conclusions: 1.Treament namely IFN-γat 500U/ml for 48 hours is optimum schemewhich induces the expression of membrane molecule MHC class I of U251 cells.2.Single ingredients namely treatments with β-elemene at 0.1mg/ml for 2 hours orheat shock at 43℃for 2 hours was optimum scheme which induces the expression ofmembrane molecule HSP 70 of U251 cells; 3.Double ingredients namely treatmentswith IFN-γat 500U/ml for 48 hours and then heat shock at 43℃for 2 hours isoptimum scheme which induces the expression of membrane molecule MHC class Iand HSP 70 of U251 cells; 4.The study suggests the membrane-enriched twomolecules namely MHC class I and HSP 70 molecule GBM U251cell vaccinepossesses high immunogenicity, the effector cells stimulated by it own higher specifickilling competence. These findings may help optimize immunotherapeutic strategy forthe treatment of malignant gliomas.