Gene therapy and non-invasive imaging method for upper gastrointestinal cancer

胃癌腹膜转移的有效治疗尚未确立。因此，我们致力于开发可连续复制的腺病毒作为治疗这些病变的新策略。我们开发了具有抗肿瘤作用并表达荧光素酶的新载体。我们的病毒载体在缺失的E-3区具有荧光素酶表达基因，当病毒复制时，该基因被激活。E1调控的Cox 2启动子和5：3嵌合纤维在胃癌细胞中表现出良好的特异性复制能力。腺病毒死亡蛋白保留在缺失的E-3区，以保存病毒的天然溶瘤能力。我们的病毒仅在肿瘤细胞中复制，并且可以以复制依赖的方式被检测为发光。我们直观地证实了胃癌细胞皮下肿瘤中的病毒复制。它具有溶瘤和侧向扩散能力。我们的载体可以在肿瘤细胞中复制，适用于荧光检测微小病变。另外，我们建立了表达红色荧光蛋白的胃癌细胞系和红色荧光腹膜播散模型小鼠。用荧光素酶表达腺病毒载体处理肿瘤细胞，通过荧光分析和发光分析评价病毒复制和肿瘤缩小情况，并进行体外检测，发现纤维修饰（5/3嵌合纤维）对实现早期病毒复制非常重要。腹腔镜全身生物发光成像显示，该病毒可用于直接显示腹膜播散病变（12只小鼠的189个腹膜病变），检测灵敏度为78.8%，特异性为99.3%。因此，这种成像系统和术前分期腹腔镜检查的组合可能是一个有效的诊断策略，检测腹膜播散。

The effective treatment for the peritoneal dissemination of gastric cancer is not established. So we effort to develop Conditionally replicative adenovirus as new therapeutic strategy of these lesions.We developed new vectors which show an antitumor effect and express luciferase. Our viral vectors have luciferase expression gene in deleted E-three region which is activated when the virus replicates. E1 controlled Cox2 promoter and five-to-three chimeric fibers show good specific replicative ability in gastric cancer cells. The adenovirus death protein is retained at the deleted E-three region in order to conserve the native oncolytic capability of the virus. Our virus replicates only in tumor cells and can be detected as a luminescence in a replication-dependent manner. We visually confirmed the viral replication in subcutaneous tumors of gastric cancer cells. And it has oncolytic and lateral spread capability. Our vectors can replicate in tumor cells, so they are applicable to detection of tiny lesions by luminescent assay.In addition, we established gastric cancer cell lines which express red fluorescent protein and red fluorescent peritoneal dissemination model mice. They are treated with luciferase express adenoviral vector and we can evaluate viral replication and tumor shrinkage by using fluorescent assay and luminescent assay.In vitro examination was done and we noticed that fiber modification (5/3 chimeric fiber) was very important to achieve early viral replication. Laparotomic whole body bioluminescence imaging revealed that this virus could be used to directly visualize peritoneal dissemination lesions (189 peritoneal lesions in 12 mice) with a detection sensitivity of 78.8% and specificity of 99.3%. A combination of this imaging system and preoperative staging laparoscopy could therefore be an effective diagnostic strategy for detecting peritoneal dissemination.