| Objective: Dendritic cell (DC), the most potent antigen presenting cell (APC) known, is capable of priming naive T-cells and can stimulate T-cells and B-cells. It holds the process of immune responses and plays a key role in those responses.One reason for losing effective anti-tumor immunity of tumor patients is that DC in their bodies was functional defect. It could not present tumor antigen to T-cells which could identify and kill tumor cells. Therefore, research efforts are currently focused on culturing DC in vitro, enhancing its ability to present antigen and exploiting DC vaccine-based active immunotherapy. This study is to investigate the culture method of DC from cord blood, to observe anti-endometrial cancer effect of CTL stimulated by DC vaccine which were prepared by loading DC with endometrial cancer antigen , and then to evaluate the possibility whether DC vaccine could be used to the immunotherapy of endometrial cancer.Method: Tumor lysate was made from frozen-thawed endometrial cancer cell lines Ishikawa. In vitro, the mononuclear cell isolated from cord blood (CB) was cultured and proliferated into DC by using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) , recombinant human interleukin-4 (rhIL-4) and recombinant human tumor necrosis factor-α(rhTNF-α). DC was loaded with tumor lysate to prepare DC vaccine, which used to stimulate T lymphocytes and to induce antigen-specific CTL. The process of DC's growth and the change of its morphous were observed by inverted microscope. The appearance of DC's was observed by scanning electron microscope. The changes of the expressions for surface markers on DC after it loaded tumor lysate, were performed by FACS. The ability of loaded-DC to stimulate proliferation of T lymphocytes, compared with DC and tumor layste, was determined by mixed lymphocyte reaction (MLR). The specific cytotoxicity of CTL induced by loaded-DC to Ishikawa was observed by MTT method, as compared with SKOV-3 and SMMC7721 tumor cells. Meanwhile, the DC group, tumor lysate group and lymphocyte group were also determined. The concentrations of IL-12 and IFN-r for the four groups (loaded-DCgroup, DC group, tumor lysate group and lymphocyte group) were measured in culture supernatants by ELISA.Results: 1 CB mononuclear cells could be induced into typical DC from all of the 8 samples. The DC was large and had a lot of fingered tubers. The expressions of its surface markers were respectively CD1a (45.31±7.42)%,CD83 (50.82±8.31)%,CD80 (63.04±7.90)%,CD86 (67.10±7.95)% and HLA-DR (73.45±8.95)%. It could also stimulate allogeneic T-cells. The stimulation index (SI) was 2.45±0.31. 2 For the loaded-DC, the expressions of these surface markers were respectively CD1a (53.54±6.82)%,CD83 (61.50±8.98)%,CD80 ( 82.51±7.16)%,CD86 (82.41±6.90)%, and HLA-DR (86.76±6.66)%. They were all higher than those on DC, P<0.05(Table 1). And the proliferation of allogeneic T-cells was markedly enhanced (SI=4.49±0.50) than DC (SI=2.45±0.31) and tumor lysate(SI=1.07±0.12), P<0.01(Table 2).3 In the ratio of 10:1,50:1and 100:1, the cytotoxicity rates of CTL stimulated by loaded-DC against Ishikawa were respectively (39.23±5.11)%, (67.82±7.64)% and (84.34±9.21)%. As compared, the cytotoxicity rates against SKOV-3 and SMMC7721 tumor cells were respectively (84.34±9.21)%,(25.48±5.59)%,(32.30±5.30)% and (14.15±2.91)%,(21.87±5.30)%,(28.42±4.59)%. In the same ratio, the cytotoxic effect of loaded-DC group against Ishikawa was much powerful than that against SKOV-3 and SMMC7721, P<0.05(Table 3), and it became more powerful as the ratio increasing. In the ratio of 100:1, it was most powerful (Fig 7).In the ratio of 10:1,50:1and 100:1, the cytotoxicity rates of DC group against Ishikawa were respectively (12.55±2.12)%,(24.95±4.13)% and (31.25±5.46)%. In the same ratio, the cytotoxic effect of tumor lysate group and lymphocyte group to Ishikawa were respectively (10.39±1.17)%,(15.79±3.64)%,(22.18±5.18)% and (6.96±1.30)%,(9.40±1.59)%,(10.22±2.51)%. In the same ratio, the cytotoxicity of loaded-DC group was much higher than the other groups, P<0.05(Table 4 and Fig 8).4 Concentration of IL-12 and IFN-r in loaded-DC group(228.54±33.39 pg/ml and 40.89±4.56 pg/ml)were increased more than those in DC group(129.85±21.83 pg/ml and 25.54±4.50 pg/ml), tumor-lysate group (21.15±5.14 pg/ml and 15.45±4.25 pg/ml)and lymphocyte group(6.54±2.48 pg/ml and 8.29±3.13 pg/ml) (Table 5 and Fig 9).Conclusions: 1 By using rhGM-CSF,rhIL-4 and rhTNF-α, it is possible to induce CB mononuclear cells into DC, which has a lot of fingered tubers, all positive express CD1a,CD83,CD80,CD86,HLA-DR, and could stimulate allogeneic T-cells.2 In vitro, the DC vaccine could stimulate allogeneic T-cells and could induce specific cytotoxic effect to endometrial cancer cell lines Ishikawa. This demonstrated that DC could present antigen effectively, stimulate T-cell to produce powerful immune response, which could educe specific and powerful anti-cancer effect.3 For the DC vaccine, the cytotoxicity and concentration of cytokine were all much higher than DC group, tumor lysate group and lymphocyte group. It indicated that the therapeutic effect of DC vaccine for immunotherapy would be more powerful than DC, tumor lysate vaccines and the immunoenhancer.4 This study would provid grouds for using DC vaccine-based active immunotherapy to endometrial cancer in vivo, explore the further immunotherapy method to endometrial cancer, and maybe could create a new way for the biotherapy of endometrial cancer.
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