| Dendritic cells(DC)play important roles in immune response processthrough their antigen presenting function. Differentiation of DC may bedivided into two different stages, immature DC can capture and processantigens, and once they are matured they lose their ability to capture andprocess antigens, but matured DC turn into antigen-presenting cells (APC)and can prime the native T cells, which play a key role in the primaryimmune response. DC pulsed with tumor antigens can induce anti-tumorimmune as vaccine. How to enhance the immunogenicity and presenting oftumor antigens are the important field and the basic of tumor vaccineresearch. Objectives: Mice marrow derived dendritic cells were pulsed withElemene-combo tumor cell vaccine(EC-TCV),MMC induced apoptoticHca-F cells or freeing-thawing treated necrotic Hca-F cells. Theirphenotypes, cytokine excretion, and cytotoxicity of T lymphocyte cellsagainst Hca-F were compared. Methods: Femurs and tibiae of mice were removed and skins andsurrounding muscles were stripped, marrow cells were collected andwashed three times with phosphate buffer solution. Cells were cultured for6 days in RPMI 1640 medium supplemented with GM-CSF (10ng/ml) andIL-4 (10ng/ml), the nonadherent DCs were collected. DCs were pulsedwith EC-TCV, apoptotic or necrotic Hca-F cells for 24 hours. Pulsed DCswere mixed with spleen T lymphocytes and cultured for 72 hours.Phenotypes of DCs were measured by flow cytometer; the proliferation ofspleen T lymphocytes and their cytotoxicity against Hca-F were evaluatedwith MTT assay; concentration of IL-12 and IL-2 of supernatant weremeasured by ELISA. Results: 1. The changes in phenotypes of pulsed DCs : The percentage ofCD40 DCs cultured with RPMI1640 was 69.27%±1.07%.That of DCs +pulsed with EC-TCV was 91.55%±1.65%. That of DCs pulsed withapoptotic Hca-F cells was 85.93%±0.13%. That of DCs pulsed withnecrotic Hca-F cells was 72.37%±1.32%. The percentage of CD86 + DCscultured with RPMI1640 was 76.4%±0.85%.That of DCs pulsed withEC-TCV was 97.89%±0.34%. That of DCs pulsed with apoptotic Hca-Fcells was 96.9%±0.18%. That of DCs pulsed with necrotic Hca-F cellswas 95.01%±0.01%.The results showed that DC pulsed with antigensup-regulate positive percentage of CD40 and CD86, The highest of allgroups was DCs pulsed with EC-TCV. 2. The concentration of IL-12 in DC culture supernatant: IL-12concentration of supernatants of control DCs cultured with RPMI1640 was114.72±23.41 ng/ml. That of DCs pulsed with EC-TCVC was 517.52±22.59 ng/ml. That of DCs pulsed with apoptotic Hca-F cells was 494.37±26.83 ng/ml. That of DCs pulsed with necrotic Hca-F cells was 413.74±43.49 ng/ml. 3. The concentration of IL-2 in spleen T lymphocytes culturesupernatant: At 3 day: if T lymphocytes are co-cultured with control DCs itis 279.5±17.67 ng/ml, If T lymphocytes are co-cultured with DCs pulsedwith EC-TCV it is 462±35.3 ng/ml, if T lymphocytes are co-cultured withDCs pulsed with apoptotic cell it is 422±63.63 ng/ml, if T lymphocytesare co-cultured with DCs pulsed with Necrotic cell it is 402±7.07 ng/ml.At 5 day: if T lymphocytes are co-cultured with control DCs it is 249.5±74.24 ng/ml, If T lymphocytes are co-cultured with DCs pulsed withEC-TCV it is 554±45.38 ng/ml, if T lymphocytes are co-cultured withDCs pulsed with apoptotic cell it is 404.5±63.63 ng/ml, if T lymphocytesare co-cultured with DCs pulsed with Necrotic cell it is 394.5±45.96ng/ml. Results show the remarkable difference exists between antigenspresenting activity of DCs pulsed with tumor cells treated differently. Thehighest of all groups is EC-TCV+DC. 4. The cytotoxicity rate of activated T lymphocytes to HCA-F: Tumor specific cytotoxicity against Hca-F by CTL activated DC issignificantly higher than control DC. Conclusion: Our results showed that EC-TCV, apoptotic cells, andnecrotic cells can induce murine myeloid DC to high expressioncostimulatory molecules CD40 and CD86,and the pulsed DC can activateand proliferate spleen T lymphocytes。The concentration of IL-12 in culturesupernatant of pul...
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