Study Of Anti-mouse Hepatocellular Carcinoma On TIL Stimulated By DC And Superantigen SEA In Vitro

Objective : To investigate the killing activity of tumor infiltrating lymphocytes (TIL) that were stimulated by dendritic cells (DC) and superantigen (SAg) staphylococcal enterotoxin A (SEA) on mouse H22 hepatocellular carcinoma cells (H22 cells) in vitro. Further, to analyze the mechanism of killing activity and find a new effective way of adoptive immunotherapy on hepatocellular carcinoma.Methods:①mouse tumor model was generated by endermic injecting H22 cells.②TIL were isolated from the H22 tumor by Lymphocyte Separation Medium and then were cultured to amplification in RPMI1640 total culture medium with 10% calf Serum and recombined murine interleukin-2 in vitro.③mouse spleen lymphocytes were isolated from the spleen of the mouse bearing tumor and then were cultured to amplification in RPMI1640 total culture medium with 10% calf Serum and recombined human interleukin-2 in vitro.④Bone marrow cell suspensions were isolated from the mouse bearing tumor, then the granulocytes,T lymphocytes, B lymphocytes, NK cells, monocytes and macrophages were removed by treating successively with specific rat anti-mouse CD4,anti-mouse CD8a, anti-mouse CD45R monoclonal antibodies,rabbit complement buffer and by semi-adhesion of DC. DC were cultured with mouse granulocyte macrophage-colony stimulating factor (GM-CSF) and mouse interleukin-4 (IL-4), then a large number of DC were obtained and DC were identified.⑤DC were cultured and sensitized by H22 cells antigen in vitro.⑥TIL were stimulated by S-DC (DC had been sensitized by H22 cells antigen) and SEA in vitro.⑦Cytoxicity assays experiment,we divided the effector cells into eight groups:TIL were stimulated by S-DC and...