Application of tetracycline inducible promoter based CRAd to cell vehicle system

本项目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型腺病毒。选用四环素诱导型启动子，便于启动子的激活切换。用四环素可诱导启动子取代野生型EL启动子，构建了基于四环素可诱导启动子的复制型腺病毒AdTRE-E1。在四环素存在下，AdTRE-E1在感染的人成纤维细胞培养细胞(WI38)中增殖。相反，在没有四环素的情况下，病毒是沉默的。因此，在培养液中添加四环素起到了启动病毒复制的开关作用。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。将AdTRE-E1和AdTERT-E1同时感染WI38细胞，加入四环素。感染72小时后，病毒迅速增殖，破坏被感染的细胞，并释放到培养液中。将含有病毒的部分培养液转移到完整的癌细胞培养瓶中。AdTERT-E1在不含四环素的情况下感染癌细胞并在细胞内独立增殖。因此，感染的WI38细胞携带病毒在细胞内，保持了病毒的感染力。四环素诱导系统在裸鼠体内的腹膜腔起作用。将感染的WI38细胞移植到小鼠的腹膜腔内，饮用饮用水中的四环素后，病毒在腹膜腔内增殖。因此，该系统在体内和体外都能发挥作用，为病毒载体的细胞载体系统提供了可能。

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.