融合基因mGM-CSF/hIL-2在肝癌细胞瘤苗特异性免疫治疗中的作用

Application of Fusion Gene mGM-CSF/hIL-2 to Specific Immunotherapy Induced by Hepatic Tumor Cell Vaccine

背景与目的:手术切除是治疗肝癌的主要方法,但对其术后的复发转移却无更好的办法。近年来,免疫学的发展使肝癌的治疗有了更好的治疗方法。本研究旨在通过制备人白细胞介素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肝癌瘤苗,观察其特异性抗肿瘤免疫作用。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细胞,制成疫苗,皮下接种小鼠,同时建立荷瘤小鼠模型。用51Cr释放法测定瘤苗免疫组、空载组、未转染组小鼠脾细胞对亲本H22细胞的杀伤活性。取血检测血清中IL-10、IFN-γ水平,观察小鼠存活期。结果:成功制备了含有hIL-2与mGM-CSF融合基因的H22肝癌瘤苗。免疫小鼠脾细胞体外对H22细胞的杀伤率为38.3%,显著高于对S180细胞的9.1%,以及空载组和未转染组的13.6%和7.5%(P<0.05)。转基因瘤苗免疫组血清IFN-γ为(12.83±0.75)pg/mL,较空载瘤苗组的(7.83±0.65)pg/mL明显升高(P<0.01),血清IL-10[(4.58±0.34)pg/mL]较空载瘤苗组的(8.15±0.28)pg/mL明显降低(P<0.01)。同时,转基因瘤苗免疫组小鼠存活期为(40±6)d,较对照组[空载瘤苗组(30±3)d,未转染组(19±4)d]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,延长荷瘤小鼠存活期。

BACKGROUND ＆ OBJECTIVE： Surgical resection is the main treatment for primary liver cancer, but there is no ideal treatment for postoperative recurrence and metastasis. Recently, due to the development of immunology, immunotherapy is regarded as a promising treatment for hepatoma. This study was to prepare H22 hepatoma tumor vaccine modified by the fusion gene of mouse granulocyte-monocyte colony stimulating factor （mGM-CSF） and human interleukin-2 （hIL-2）, and explore its specific antitumor immunity. METHODS： Eukaryotic vector expressing fusion gene mGM-CSF/hIL-2 was transfected into H22 cells to establish tumor vaccine （H22/GM-CSF/IL-2） and inoculate mice; pcDNA3.1 was also transfected into H22 cells as empty control （H22/neo）. Tumor-bearing mouse models were established. The cytotoxicities of the splenocytes from the mice in H22/ GM-CSF/IL-2, H22/neo, and blank control groups were examined by ^51Cr release assay. The serum levels of interleukin-10 （IL-10）, interferon-γ （IFN-γ） were detected by ELISA. The survival of mice was observed. RESULTS： H22 cell vaccine modified by mGM-CSF/hIL-2 was successfully established. The killing rate of H22 cells by splenocytes was significantly higher in H22/GM- CSF/IL-2 group than in H22/neo and blank control groups （38.3% vs. 13.6% and 7.5%, P〈0.05）. The serum level of IFN-γ was significantly higher and the serum level of IL-2 was significantly lower in H22/GM-CSF/IL-2 group than in H22/neo group [（12.83±0.75） pg/mL vs. （7.83±0.65） pg/mL, P〈 0.01; （4.58±0.34） pg/ml vs. （8.15±0.28） pg/mL, P〈0.01]. The survival time of mice was significantly longer in H22/GM-CSF/IL-2 group than in H22/ neo and blank control groups [（40±6） days vs. （30±3） days and （19±4） days, P〈0.01]. CONCLUSION： Transfecting fusion gene mGM-CSF/hIL-2 into the novel autologous tumor vaccine H22 can elicit specific cellular immune response and improve the host＇s antitumor immune response, and prolong the survival of tumor-bearing mice.