Gene therapy for cancer using the gone gun system

为了建立无载体的基因治疗系统，我们对基因枪的基因转导效果进行了检测。虽然我们证实了LacZ基因在胰腺癌细胞中的表达，但转导效率很低。对于基因枪系统来说，选择合适的基因来表达低浓度的高效抗肿瘤作用是非常重要的。作为一种抗肿瘤药物，IL-12被认为是免疫应答的关键调节因子，尤其是涉及CTL和NK细胞的免疫应答。在此，我们报道了IL-12对NK耗竭的SCID小鼠的人和小鼠肿瘤的抗血管生成作用，使用的是通过基因工程产生这种细胞因子的成纤维细胞。尽管IL-12的存在对肿瘤细胞的体外生长没有影响，但共接种分泌IL-12的成纤维细胞强烈地抑制了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，肿瘤周围的新生血管明显受到抑制，从而抑制了肿瘤的生长。肿瘤标本切片中的凝集素染色也显示血管数量显著减少。IL-12刺激内皮细胞表达干扰素-γ及其诱导的抗血管生成趋化因子IP-10RNA。IL-12还下调内皮细胞有丝分裂原血管内皮生长因子和碱性成纤维细胞生长因子的表达。IL-12的抗肿瘤作用伴随着有趣的组织学变化，包括高度的角化和凋亡，人类肿瘤的增殖率下降和小鼠肿瘤的广泛坏死。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.