Application of tetracycline inducible promoter based CRAd to cell vehicle system

本课题的目的是建立病毒载体的细胞载体系统。为此，首先制备了基于药物诱导启动子的条件复制腺病毒。选择四环素诱导型启动子以容易地切换启动子激活。用四环素诱导型启动子替换野生型E1启动子，构建了四环素诱导型腺病毒AdTRE-E1。在培养基中存在四环素的情况下，AdTRE-E1在感染的人成纤维细胞培养细胞（WI 38）中增殖。相反，在没有四环素的情况下，病毒是沉默的。因此，在培养基中添加四环素可作为病毒复制启动的开关。在下一步中，基于端粒酶启动子的复制型腺病毒（AdTERT-E1）与四环素诱导系统一起用于癌症治疗。同时用AdTRE-E1和AdTERT-E1共感染WI 38细胞，并在培养液中加入四环素。在感染72小时后，病毒迅速增殖并破坏感染的细胞并释放到培养基中。将一部分含有病毒的培养基转移到完整的癌细胞培养瓶中。AdTERT-E1感染癌细胞并在不含四环素的细胞中独立增殖。结果，感染的WI 38细胞将病毒携带在细胞中，保持了病毒的感染能力。四环素诱导系统在裸鼠腹腔内发挥作用。当将感染的WI 38细胞植入小鼠腹膜内时，在饮用水中喂食四环素后，病毒在腹膜间隙中增殖。因此，该系统在体内和体外均能发挥作用，并有可能作为病毒载体的细胞载体系统。

The aim of this project is to establish the cell vehicle system for the viral vector carrier. To do this, drug inducible promoter based conditionally replicative adenovirus was made at first. Tetracycline inducible promoter was chosen to switch the promoter activation easily. The tetracycline inducible promoter based replicative adenovirus, AdTRE-E1, was constructed by the replacement of wild type El promoter with tetracycline inducible promoter. AdTRE-E1 proliferated in the infected human fibroblast culture cells (WI38) in the presence of tetracycline in the culture medium. Contrary, the virus was silent in the absence of tetracycline. Therefore, addition of tetracycline in the medium functioned as a switch for viral replication initiation. In the next step, telomerase promoter based replicative adenovirus (AdTERT-E1) was used with the tetracycline inducible system for cancer treatment. WI38 cells were co-infected with AdTRE-E1 and AdTERT-E1 simultaneously, and then tetracycline was added into the culture medium. After 72 hours from infection, the viruses proliferated rapidly and destroyed the infected cells and were released into the medium. Apart of the medium containing viruses was transferred to the intact cancer cell culture flask. AdTERT-E1 infected into the cancer cells and proliferated in the cells independently without tetracycline. As a result, infected WI38 cells carried the viruses in the cells keeping the virus ability of its infectivity. The tetracycline inducible system functioned in the peritoneal space in the nude mouse. When the infected WI38 cells were implanted into the mouse intraperitoneally, viruses proliferated in the peritoneal space after feeding of tetracycline in the drinking water. Therefore, this system functioned in vivo as well as in vitro, and was possible for the cell vehicle system for virus carrier.