Purification Of HSP70-peptide Complex And Plused DC Induces Antitumor Effect In Vitro

Objective To establish a new method of purifying HSP70-peptide complex from tumor cells lines and investigate the antitumor effect of dendritic cells ( DCs ) plused by HSP70-peptide complex in vitro.Methods Cytosolic HSP70-peptide complex of heat treated SGC was purified successively by chromalographic procedures.including heparin-sepharose chromatographic column. DEAE-sepharose chromatographie column and ADP-agarose chromatographic column.The molecular weight and identity of protein obtainded was confirmed by SDS-PAGE and Westernblot. The final protein was quantified with Bradford assay.DCs were cultured from peripheral blood mononuclear cells with medium including granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4(IL-4). then pulsed by HSP70-peptide complex. The phenotypes of those were analyzed by flow cytometry. Lymphocytes were stimulated by DCs plused with HSP70-peptide complex. Proliferation activity of Lymphocyte and Lymphocyte cytoloxicily were measured by MTT.Results A sharp stained protein band with a molecular weight of 72KD was obtained and shown to be HSP70-peptide complex as confirmed by Westernblot. The final protein recovery was 56.36ug per 1g tumor cells(wet weight). DCs could be obtained from peripheral blood mononuclear cells with GM-CSF and IL-4 and become mature with the effect of HSP70-peptide complex.These mature DCs highly expressed HLA-DR. CD83 and CD86.lymphocyte activated by this mature DCs could kill SCG specifieitily .but not BGC and CW-2.Conclusions The high-purity HSP70 with message of tumor antigen could be obtained by using chromatographic procedures with heparin-scpharose chromatographic column. DEAE-sepharose chromatographic column and ADP-agarose chromatographic column. DCs modified by HSP70-peptide complex...