| Background&Objective: DC based anti-tumor immunotherapy has become the research highlight. It was found that cancer cells share several oncofetal antigens or other cell surface molecules with embryonic stem cells. Our research fabricated embryonic stem cell lysate antigens and used the antigens sensitized dendritic cells to make DC vaccines, aiming to observe the anti-tumor immunotherapeutic effect on the transplantable subcutaneous tumor model of murine hepatic tumor.Methods: 1. C57BL/6J mouse bone marrow derived dendritic cells were cultured by cytokines(rm GM-CSF、rm IL-4), which were induced to mature by embryonic stem cell or Hepa1-6 lysate antigens to make DC vaccines. The condition of different antigen sensitized DC vaccine expressing CD11 c, CD80, CD86 were analyzed by flow cytometry; 2.Mouse hepatoma cell line was injected into mouse abdomen subcutaneously to construct a transplantable subcutaneous tumor model of murine hepatic tumor. According to DC pulsed, groups were divided into ESC-DC vaccines group、Hepa1-6-DC vaccine group、DC vaccine group and Control group(PBS group), with 10 mice in each group. vaccining three times at an one-week interval respectively. Then we measured the tumor length and diameter every five days. The endpoint for this study was one tumor diameter ≥ 15 mm or earlier if tumor ulcerated, at which point mice were euthanized and tumors were weighed. Five days after the completing treatment, ELISA assay was used to detect the Ig G levels in mice peripheral blood; LDH cytotoxicity assay was used to detect the cytotoxicity of mice spleen lymphocyte; the CD4+CD25+Fox P3+ T cell(treg) content in mice spleen lymphocyte was analyzed by flow cytometry.Result: 1. We had acheived the success in culturing bone marrow derived dendritic cells. By analysing the phenotype of DC, the surface molecular CD11 c accounted for about 78%, the amount of suffer molecular(CD80, CD86) in ESC-DC was higher than Hepa1-6-DC, DC(p<0.05); 2. During the therapy, we measured the tumor volume. Comparison with the control group PBS group, the average growth of subcutaneous hepatoma were significantly reduced in ESC-DC vaccination group、Hepa1-6-DC vaccination group、DC vaccination group. The average weight of tumors were 0.1573 gram(g) in ESC-DC vaccination group, 0.2387 g in Hepa1-6-DC vaccination group, 0.2620 g in DC vaccination group, 0.3633 g in control group; 3. Five days after the completing treatment, ELISA assay was found: the levelof Ig G in peripheral blood in ESC-DC vaccine group mice was higher than Hepa1-6-DC vaccination group、DC vaccination group and PBS group, the average value were 77.99 ± 6.43ng/ml, 49.47 ± 4.36ng/ml, 40.61 ± 5.17ng/ml, 17.21 ± 2.55ng/ml, respectively; the cytotoxicity of mice spleen lymphocyte: ESC-DC vaccine group>Hepa1-6-DC vaccine group>DC vaccine group>PBS group; flow cytometry analyzed the amount of CD4+CD25+Fox P3+T cell in mice spleen and it was found that the amount of Treg in ESC-DC vaccine group was obviously lower than other three groups(p<0.05).Conclusion: 1. In vitro, Embryonic stem cell lysis antigen can effectively induce C57BL/6J mouse bone marrow derived dendritic cells to matrue, and the effect is superior to that of mouse hepatoma cell line(Hepa1-6) lysis antigen; 2. Both ESC-DC vaccine and Hepa1-6-DC vaccine are able to induce anti-tumor immunotherapeutic effect on subcutaneous implantable hepatoma, slow the average growth of subcutaneous hepatoma; 3. Comparison with Hepa1-6-DC vaccine、DC vaccine, the anti-tumor immune effect of ESC-DC vaccine is more excellent, for the clinical application of DC vaccines provide the theoretical and experimental basis. |
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