The Effect Of DC From Cord Blood CD34～+ Cell On Human Hepatoma Cell BEL-7402 In Vitro And In Vivo

Objective To prepare cancer vaccine of dendritic cells (DCs) derived from human cord blood using whole tumor lysates and study its cytotoxic activity on human hepatoma cells in vitro and in vivo.Methods CD34+ hematopoietic stem cells were isolated from human cord blood mononuclear cells with CD34+ progenitor cell isolation kit by Mini MACS(magnetic activated cell sorting), and then stimulated with 100 g/ml recombined human granulocyte-macrophage colony-stimulating factor(rhGM-CSF) and 50U/ml recombined human tumor necrosis factor (rhTNF)- for two weeks. The differentiation process of DC was observed under the phase contrast microscope. Allogeneic lymphocytes from peripheral blood or cord blood were primed into cytotoxic T lymphocytes(CTLs) by DCs pulsed with supersonically lysed human hepatoma cell BEL-7402.The cytotoxic activity of CTL in vitro was detected by live cell counting and Neutral red uptake assay. For study the effect of CTL on hepatoma cell in vivo, SCID mice were divided into treatment group and immunity group. The mice in treatment group were established human hepatoma model by subcutaneous injection of BEL-7402 2 106 in the right flank. Subsequently, the tumor-bearing mice were injected subcutaneously with 1 107 CTLs primed by DC vaccine or unprimedlymphocytes from peripheral blood, twice at interval of 3 days. The mice in immunity group were first immunized with 1 X 107 CTLs or unprimed lymphocytes twice. 4 days after last immunity, the mice were challenged with 2 106hepatoma cells BEL-7402. 32 days after tumor cells injection, all of the mice were killed.Results Under the stimulation of rhGM-CSF and rhTNF-a, CD34+ cells isolated from cord blood could proliferate and form cell clones. The morphology of cell changed by displaying cytoplasm processes during the differentiation. In later period of the culture, DCs shed from the clones to the medium. DCs pulsed with supersonically lysed BEL-7402 stretched out more pronounced dendritic processes. Counting of live cells and assaying of neutral red uptaking indicated that the cytotoxicity of CTL primed by cord blood derived-DC vaccine was significantly higher than of unprimed lymphocytes from both peripheral blood and cord blood in vitro (P<0.01). There was no significant difference between the cytotoxic activity of CTL from cord blood and that from peripheral blood (P>0.05). In tumor-bearing mice treated with CTLs, the growth speed of tumor decreased, and the tumor size and tumor weight were less than treated with unprimed lymphocytes (P<0.05). Compared with mice immunized with unprimed lymphocytes or mice treated with CTLs, SCID mice immunized with DC vaccine primed CTLs were with low tumor incidence (P<0.05), delayed tumor latent period (P<0.01) and small tumor volume (P<0.05).Conclusions The percentage of CD34+ cells in cord blood was more than in peripheral blood. CD34+ cells from cord blood could expand and differentiate into DC under the stimulation of rhGM-CSF and rhTNF-a. Pulsing DC with whole tumor lysates led DC come into mature stage with pronounced dendritic processes. Whole tumor lysates Pulsed DCs werecapable of activating lymphocytes from peripheral blood as well as naive lymphocytes from cord blood. Cord blood derived-DC vaccine had the activity of inhibiting growth of human hepetoma cells in vitro and in vivo. It was indicated that DC vaccine was with immunotherapeutic effect and could protect body from tumor challenge. The immunoprotective effect of DC vaccine on human hepatoma was more potent than immunotherapic effect.