融合基因GM-CSF/IL-2转基因瘤苗在肝癌特异性免疫治疗中的应用

Application of a tumor cell vaccine transfected with GM-CSF/IL-2 fusion gene for specific immunotherapy of hepatocellular carcinoma

目的制备人白细胞介素2(hIL-2)与鼠粒-单核细胞集落刺激因子(mGM-CSF)融合基因修饰的H22肝癌全细胞瘤苗,探索融合基因GM-CSF/IL-2转基因瘤苗,在肝癌主动免疫治疗中特异性抗肿瘤作用。方法用含hIL-2与mGM-CSF融合基因的真核表达载体在体外转染H22肝癌细胞,制成疫苗,皮下接种Balb/c小鼠,同时建立Balb/c小鼠荷瘤模型,ELISA法检测H22/pcDNA3.1(+)-GM-CSF/IL-2瘤苗免疫组小鼠与各对照组小鼠(分别接种H22/pcDNA3.1(+)瘤苗、H22瘤苗、PBS)血清中IL-10、IFN-γ水平,观察小鼠存活期;同时用51Cr释放法测定H22/pcDNA3.1(+)-GM-CSF/IL-2瘤苗免疫组小鼠与各对照组小鼠(单纯荷瘤组、正常组)脾细胞对亲本H22肝癌细胞的杀伤活性。结果成功制备了含有hIL-2与mGM-CSF融合基因的H22肝癌瘤苗,转基因瘤苗免疫组小鼠的脾细胞在体外对亲本H22肝癌细胞的杀伤率为38.3%,显著高于对照组小鼠的脾细胞对亲本H22肝癌细胞的杀伤率(分别为13.6%和7.5%),也高于其对S180细胞的杀伤率(9.1%)(P<0.05)。转基因瘤苗免疫组小鼠血清IFN-γ较对照组明显升高(P<0.01),血清IL-10较对照组明显降低(P<0.01)。同时,转基因瘤苗免疫组小鼠生存期亦有明显延长。结论转染hIL-2与mGM-CSF融合基因的同系肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应,延长荷瘤小鼠生存期。

Objective To prepare a transgenic tumor cell vaccine transfected the fusion gene ofmurine granulocyte-monocyte colony stimulating factor （mGM-CSF） and human interleukin-2 （hlL-2） using H22 cells, and explore its specific antitumor immunity against hepatocellular carcinoma. Methods The eukaryotic vector expressing the fusion gene mGM-CSF/hlL-2 was transfected into H22 cells followed by radiation exposure to construct the tumor cell vaccine, which was used to immunize Balb/c mice by subcutaneous inoculation. The mice inoculated subcutaneously with H22 cells, cells transfected with the empty vector pcDNA（＋）, or with PBS served as the controls. A week later, H22 cells were injected peritoneally into Balb/c mice for establishing the tumor-bearing model, and their serum levels of interleukin-10 （IL-10） and interferon-γ（IFN-γ） were detected using enzyme-linked immunosorbent assay （ELISA） with the survival of the mice recorded. The spleen cells were obtained from the mice immunized with the tumor cell vaccine, the tumor-beating mice and the normal control mice to assess their cytotoxicity against the parental H22 cells in vitro using 51C：release assay. Results The transgenic H22 cell vaccine transfected with mGM-CSF/hlL-2 fusion gene was successfully constructed. The killing rate of H22 cells by the spleen cells from the mice immunized with the transgenic cell vaccine was significantly higher than those by the spleen cells from the tumor-bearing mice or normal control mice （38.3% vs 13.6% and 7.5%, P〈0.05）. Serum IFN-γ in the tumor-bearing mice immunized with the transgenic cell vaccine was significantly higher, and serum IL- 10 significantly lower than those of the control groups （P〈0.01）. The survival time of the tumor-bearing mice injected with the transgenic cell vaccine was also significantly prolonged. Conclusion Syngeneic tumor cell vaccine genetically modified by mGM-CSF/hIL-2 fusion gene transfection can elicit specific cellular immune response and enhance the host antitumor immune response to extend the survival time of tumor-beating mice.