| ObjectiveBased on previous significant findings, this study was designed to investigate the effects of heat shock protein (HSP) 70-peptide complex, which was purified from H22 hepatic carcinoma treated with high intensity focused ultrasound (HIFU), on host anti-tumor immunity in mice. HSP70-peptide complex was used as a tumor antigen to activate immature dendritic cells (DCs) in vitro. Then large amounts of cytotoxic T lymphocytes (CTL) were obtained by stimulating T lymphocytes with activated DCs, and CTL specific cytotoxicity against H22 tumor cells was measured in vitro. Methods1. Extraction of myeloid DCs in mice Myelocytes derived from normal mice were cultured with rmIL-4 and rmGM-CSF. Cellular characteristics and cell growth were observed during the culture. Using fluorescein activated cell sorter (FACS),the expression of CD11c and DEC 205 on the cell membrane was measured.2. Activation of DCs with tumor antigens Antigens used for activation of DCs were classified as following groups: B16 HSP70-peptide complex group, the HSP70 was prepared from HIFU-treated B16 tumor; H22 treated-tumor supernatant group, the tumor antigen was from HIFU-treated H22 tumor; H22 tumor supernatant group, the antgen was from H22 tumors; HSP70-peptide complex group, the tumor antigen purified from HIFU-treated H22 tumor; serum group, the antigen was from mouse serum. The antigens were respectively added into the culture for 24-hour co-culture with DCs. Using FACS the expression of CD80, CD86 and MHCII was measured in each group for evaluation of activated DCs. The level of IL-12 from mature DCs and INF-γsecreted by T lymphocytes was assessed using ELISA technique.3. Anti-tumor immunity induced by activated DCs DCs activated by the antigens in each group were employed to stimulate spleen lymphocytes, and MTT assay was performed to assess spleen lymphocyte proliferating activity. CTL specific cytotoxicity against H22 tumor cells were measured using MTT assay 4. SPSS10.0 software was used for statistical analysis. Results1. Extraction of myeloid DCs in mice Myelocytes were cultured with rmIL-4 and rmGM-CSF for 7 days, and morphological characteristics of DCs were observed. Using FACS the expression of CD11c and DEC205 on the membrane of these cells was detected. 2. Activation of DCs with tumor antigens The expression of CD80, CD86 and MHCII, and the level of IL-12 and INF-γ were increased in H22 tumor supernatant group, H22 treated tumor supernatant group, B16 HSP70-peptide complex group and H22 HSP70-peptide complex group respectively. Compared to serum group, statistical difference was significantly observed in these four groups, P < 0.001. These data were significant higher in H22 HSP70-peptide complex group than those in H22 tumor supernatant group and H22 treated tumor supernatant group (P < 0.05), but no significant difference was observed between H22 HSP70-peptide complex group and B16 HSP70-peptide complex group.3. Anti-tumor immunity induced by activated DCsSpleen lymphocyte proliferation rate was significantly higher in H22 HSP70-peptide complex group and B16 HSP70-peptide complex group than one in serum group, H22 tumor supernatant group and H22 treated tumor supernatant group (P < 0.001); but no significant difference was observed between B16 HSP70-peptide complex group and HSP70-peptide complex group(P ﹥ 0.05). CTL cytotoxicity against H22 tumor cells was 70.0%, which was significantly higher than one in serum group, H22 tumor supernatant group and H22 treated tumor supernatant group (P < 0.001). But no statistical difference was observed in CTL cytotoxicity against B16 tumor cells among these four groups. CTL cytotoxicity against B16 tumor cells was 78.5%, and the cytotoxicity against H22 tumor cells was 21.4% in B16 HSP70-peptide complex group, indicating that CTL cytotoxicity original from HIFU-treated tumor was specific anti-tumor immunity.Conclusion1. DCs obtained from myelocytes have morphological characteris... |
PDF Full Text Request