The Bioactivity Change Of Mice Marrow-Derived Dendritic Cells Pulsed With H_TA-HSP70_BCG

Dendritic cells (DC)play important roles in the process of antigen presenting.Differentiation of DC contains two stages,immature DC can capture and process antigens,once they are mature,they lose their ability to capture and process antigens,but mature DC turn into antigen-presenting cells and can prime the native T cells .The peptide binding C-terinal portion of heat shock protein HSP70 ( aa 359-610 ) induce DC to mature and induce the production of IL-12,TNF-α,NO,C-C chemokines ,etc. Our previous study showed that immunize mice with complex antigens of HCa-F elemene combo-tumor cell vaccine and HSP70 of BCG(HTA-HSP70BCG)could induce potent immune protective effects against HCa-F in mice.Objectives: In our study,we investigate the HTA-BCGHSP70 compound tumor vaccine pulse mice marrow derived DC in vitro,detect the change of DC's antigen capture and present ability .To study the bioactivity changes of DC pulsed with HTA-HSP70BCG.Methods: Femurs and tibiae of mice were removed and purified from the surrounding muscle tissue, then intact bones were washed with PBS.Cell culture medium was RPMIRPMI-1640 supplemented with GM-CSF (10ug/ml) and IL-4(10ug/ml), nonadherent murine bone marrow cells were collected the on day 6, Then DC were stimulated with HTA,HSP70BCG,HTA-HSP70BCG for 48h,Supernatant from the DC cultures was harvested on day 8,and IL-12 and NO level were measured respectively by ELISA and NO reagent box . Detect the phagocytosis FITC labeled dextran capacity of DC flow cytometer. Detect the proliferation of nonadherent splenic cells stimulated by distinct pulsed DC, the inhibitation rate of HCa-F and the cytotoxicity ability of activated nonadherent splenocytes.Results: 1 Endocytosis Percentage of FITC-labeled dextran by DC: the endocytosis percentage of pulsed DC lower than DC alone; the percentage of DC alone is 96.77%, the percentage of DC pulsed with HTA is 69.89%,the percentage of DC pulsed with HSP70BCG is 63.89%, and the group pulsed with HTA-HSP70BCG is 61.05%.The difference between each group is distinct (P<0.05) except that difference between the group of HSP70BCG and HTA-HSP70BCG is not distinct (P<0.05).The endocytosis percentage of DC alone is the highest,the percentage of HTA-HSP70BCG is the lowest.2 Different pulsed DC inhibit the proliferation of HCa-F:the inhibition rate of HCa-F by pulsed DC is higher than DC alone.According to the ratio of effective cells to target cells is 1:1, 5:1,10:1,the result of inhibition rate is DC< HTApulsed DC is higher than those activated by sole DC. According to the ratio of effective cells to target cells is 1:1, 5:1,10:1,the result of cytotoxicity rate is DC< HTA