Gene therapy for cancer using the gone gun system

为了建立无载体的基因治疗系统，我们检测了基因枪的基因转导效果。虽然我们可以证实LacZ基因在胰腺癌细胞中的表达，但转导效率很低。基因枪系统的关键是选择合适的基因，使其在低浓度下就能表达高效的抗肿瘤作用。白细胞介素-12作为一种抗肿瘤剂，已被发现是免疫应答的关键调节剂，特别是涉及CTL和NK细胞的免疫应答。我们在此报告了IL-12对NK耗竭的SCID小鼠中的人以及鼠肿瘤的抗血管生成作用，使用经基因工程改造以分泌这种细胞因子的成纤维细胞。虽然肿瘤细胞的体外生长不受IL-12的存在的影响，IL-12分泌成纤维细胞的共接种强烈抑制免疫缺陷小鼠的肿瘤生长。在植入分泌IL-12的成纤维细胞的区域中，肿瘤周围的新血管形成被显著抑制，导致肿瘤生长受到抑制。肿瘤样品切片中的凝集素染色也显示血管数量显著减少。在用IL-12培养的内皮细胞中，IFN-γ及其诱导的抗血管生成趋化因子IP-10的RNA表达被刺激。IL-12可下调血管内皮细胞有丝分裂原VEGF和bFGF的表达，并可引起肿瘤细胞高度角化和凋亡，抑制肿瘤细胞增殖，抑制肿瘤细胞坏死。因此，IL-12是该基因枪系统的候选基因。

In order to establish a gene therapy system without vectors, we checked the efficacy of gene gun for gene transduction. Although we could confirm the expression of LacZ gene in pancreatic cancer cells, the transduction efficacy was very low. It is important to choose the suitable gene which can express the efficient anti-tumor effect with low concentration for gene gun System. As an anti-tumor agent, Interleukin-12 has been revealed to be a key regulator of the immune response, particularly that involving CTL and NK cells. We report herein the anti-angiogenesis effect of IL-12 on human as well as murine tumors in NK-depleted SCID mice using fibroblasts genetically engineered to secrete this cytokine. Although the in vitro growth of tumor cells was not affected by the presence of IL-12, co-inoculation of IL-12 secreting fibroblasts strongly inhibited tumor growth in immunodeficient mice. The neovascularization surrounding the tumor was remarkably inhibited in the area where the IL-12 secreting fibroblasts were implanted, resulting in the suppression of tumor growth. Lectin staining in tumor sample sections also showed a significant reduction in the number of vessels. The RNA expression of IFN-γ and its inducible antiangiogenic chemokine IP-10 was stimulated in endothelial cells cultured with IL-12. It was also found that IL-12 downregulated the expression of the endothelial cell mitogens VEGF and bFGF.The anti-tumor effects of IL-12 were accompanied by interesting histological changes consisting of a high degree of keratinization and apoptosis and a decrease in the proliferation rate of human tumors and extensive necrosis in the murine ones. Therefore, IL-12 is the candidate gene for this gene gun system.