ABSTRACT
OBJECTIVE@#To assess the activation function of specific tumor polypeptide for dendritic cell vaccine on lymphocytes proliferation, production of cytokines and killing activity in vitro by using dendritic cells as antigen presenting vector.@*METHODS@#Peripheral blood dendritic cells (DC) and cytokine-induced killer (CIK) were isolated and cultured by adherent culture method; CCK-8 method was used to assess the proliferation function of lymphocytes and the killing function of lymphocytes to tumor cells; enzyme-linked immunospot assay method was used to evaluate the secretion function of cytokines. The experiment was divided into tumor polypeptide group (peptide with DC-CIK), DC-CIK group and CIK group.@*RESULTS@#With presence of interleukin-2 (IL-2) in the culture system, the lymphocyte proliferation of the three groups was obvious. The absorbance at 450 nm of tumor polypeptide group was significantly higher than that of CIK group at the time points day 4 and day 6 (day 4: Z=-3.79, P < 0.001; day 6: Z =-2.95, P < 0.01). The absorbance at 450 nm of group tumor polypeptide was significantly higher than that of DC-CIK group on day 4 (Z=-2.02, P < 0.05). Without IL-2 in the culture system, lymphocytes proliferated slowly in all the three groups, and there was no significant difference in 450 nm absorbance at each time point. The levels of IL-4 (Z=-2.61, P < 0.01), granulocyte-macrophage colony-stimulation factor (GM-CSF, Z=-3.85, P < 0.001), interferon- γ (IFN- γ, Z=-3.56, P < 0.001) and tumor necrosis factor-α (TNF-ɑ, Z=-3.40, P < 0.001) of tumor polypeptide group were higher than those of CIK group. There was no significant difference in the production of cytokines except IL-4 (Z=-2.15, P < 0.05) when tumor polypeptide group was compared with DC-CIK group. The production of IFN-γ (Z=-2.44, P < 0.05), TNF-ɑ (Z=-2.26, P < 0.05) and GM-CSF (Z=-3.73, P < 0.001) in DC-CIK group were higher than those of CIK group. Although there was no significant difference in killing activity between tumor polypeptide group, DC-CIK group and CIK group at hour 18 and hour 24, and the killing activity of tumor polypeptide group was higher than that of the other two groups.@*CONCLUSION@#Tumor peptide combined with dendritic cells can improve the proliferation activity of CIK cells in vitro, and increase the secretion of several cytokines.
Subject(s)
Dendritic Cells , PeptidesABSTRACT
In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.