| OBJECTIVE: To establish a new method of purifying HSP70 peptide complex (HSP70-PC) from pancreatic cancer tumor and investigate the strategies and methods of pancreatic cancer immunotherapy adopt cytotoxic T lymphocytes activated by dendritic cells (DCs), that modified by HSP70-PC.METHOD: HSP70 peptide complex from lumps of tumor-bearing mouse innoculated with pancreas cancer cell (MPC83) were purified by ConA-Sepharose and ADP-Agarose affinity chromatography. The protein of HSP70 peptide complex was quantified by SDS-PAGE and Bradford assay. Dendritic cells derived from normal murine bone marrow are induced by GM-CSF and IL-4．MTT method was applied to analyses the proliferation activities of DCs. To achieve the specific T lymphocyte cells, DCs modified with HSP70 peptide complex were coculture with murine spleen lymphocytes for 48 hours, meanwhile lymphocytes respectively stimulated by DCs modified with lysate of MPC83 and DCs without peptide as control groups. Then these activated lymphocytes were cocultured with MPC83 at ratio of effect-target 10:1 and 20:1 in vitro and the kill activities were detected by MTT method.RESULT: High purify HSP70-PC was obtained, 100μg of HSP70-PC peptide complex can be extracted from per 1g lump; DCs obtained from murine bone marrow cells expressed highly CD11c, CD80 after inducing with GM-CSF and IL-4; 10~6DCs need 1μg of HSP70-PC to modify; T cells stimulated by DCs modified with HSP70-PC show more specific cytotoxic activity to MPC83 than lymphocytes activated by DCs with lysate of MPC83 and DCs without HSP70-PC in vitro. CONCLUSION: We can extract high purity HSP70-PC for clinical individual immunotherapy from pancreatic cancer lump by using hypobaric affinity chromatography; DCs vaccine modified with HSP70-PC has significant kill effect against pancreatic cancer cell in vitro.
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