Gene therapy by tumor cells transfected with genes of costimulatory molecular and cytokines

在本研究中，我们利用腺病毒载体评估了转染IL-2、GM-CSF或共刺激分子B7基因的肿瘤疫苗的抗肿瘤免疫作用。此外，用ifn - γ培养肿瘤细胞以增加MHC I类抗原的表达。Western blot和显微荧光分析检测重组腺病毒感染细胞中IL-2和GM-CSF蛋白的表达。ELISA法检测细胞中这些细胞因子的分泌。流式细胞术分析发现，重组腺病毒转导B7基因感染后，细胞表面B7蛋白表达量极高，ifn - γ预培养后，细胞表面MHC I类抗原表达量增加。动物实验将小鼠分为7组：(1)IL2+B7+ ifn - γ (2) IL2+B7 (3) IL2 (4) B7 (5) ifn - γ(6)非转导肿瘤细胞(7)PBS。在肿瘤生长预防模型（第7天肿瘤疫苗）中，与未转导的肿瘤细胞和PBS组相比，IL2+B7+ ifn - γ、IL2+B7和IL2组肿瘤体积显著减少（P<0.01），存活率分别为50%、37.5%和25%。在肿瘤治疗模型（第1天肿瘤疫苗）中，IL2+B7+ ifn - γ组和IL2+B7组肿瘤体积显著减小（P<0.01），存活率分别为25%和12.5%。此外，IL2+B7+ ifn - γ、IL2+B7和IL2组肺转移灶数量均显著减少（P<0.01）。上述结果表明，转染IL-2和B7基因的辐照肿瘤细胞接种和IFN-r预处理对小鼠的抗肿瘤作用最强。

In this study, we evaluated the antitumor immunity of tumor vaccine transfected IL-2, GM-CSF,or costimulatory molecule B7 gene using the adenovirus vector. Furthermore, tumor cells were cultured with IFN-gamma to increase the expression of MHC class I antigen.The expressions of IL-2 and GM-CSF protein in the cells infected with recombinant adenovirus were examined by Western blot and microscopic fluorecent analysis. The secretion of these cytokines from the cells was detected by ELISA assay. From the flow cytometry analysis, the cell surface expression of B7 protein was extremely high due to the infection of recombinant adenovirus transduced B7 gene, and the expression of MHC class I antigen of cell surface increased due to the preculture with IFN-gamma . For the animal study, mice were divided into 7 groups as follows : (1) IL2+B7+IFN-gamma (2) IL2+B7 (3) IL2 (4) B7 (5) IFN-gamma (6) non-transduced tumor cells (7) PBS.In the prevention model of tumor growth (day-7 tumor vaccine), IL2+B7+IFN-gamma, IL2+B7, and IL2 groups showed the significant decreases of tumor volume as compared to the nontransduced tumor cells and PBS groups (P<0.01), and the survival rates were 50%, 37.5%, and 25%, respectively. In the therapeutic model of the tumor (day 1 tumor vaccine), IL2+B7+IFN-gamma and IL2+B7 groups showed significant decreases of tumor volume (P<0.01), and the survival rates were 25% and 12.5%, respectively. Furthermore, IL2+B7+IFN-gamma, IL2+B7 and IL2 groups showed significant deacreases in the number of lung metastases (P<0.01).These results suggests that the vaccinations of irradiated tumor cells of transfected IL-2 and B7 gene, and pretreatment with IFN-r, showed the strongest antitumor effects in mice.