腺病毒介导精氨酸脱亚氨基酶靶向性基因治疗肝癌的机制

At the forefront of medicine, Gene therapy brings us the latest research into genetic and cell-based technologies to treat disease. Cancer gene therapy is very popular recently in the field of tumor medicine, which brings us the hope that cancer would be cured thoroughly in the near future. The problems of targeting and security are not still resolved in cancer gene therapy. In our previous study, we found that human telomerase reverse transcriptase (hTERT) expresses highly in cancer cells, but keeps silence in normal cells. We used two normal cell lines (RWPE1 and L-02) to compare with four cancer cell lines (PC3, HepG2, Hep3B and Hela). The results suggest that hTERT's promoter is just activated in cancer cells. We also found that arginine deiminase (ADI) is a good anti-cancer gene. ADI's expression can induce cancer cells into apoptosis very soon, but don't affect normal cells. 30% of cell apoptosis could be checked out after ADI kept expression in Hep3B cell line for 2 days. But L-02 cell line only had less than 10% of cell apoptosis. Based on above results, we will start gene therapy research to cure hepatic tumor. pAd/PL-DESTTM adenovirus vector from Invitrogen is the best choice used for gene transfer. pAd/PL-DESTTM vector adopts GATEWAY technology which is the newest and fastest technique to insert the gene into the vector. In order to target tumor, hTERT's promoter will be used to start gene expression in adenovirus. TetO2 promoter will be also used to make gene expression regulatable in adenovirus. ADI will be the anti-cancer gene in adenovirus to keep tightly target tumor. Therefore, Promotor(hTERT)-ADI adenovirus and Promotor(TetO2)-ADI adenovirus will be constructed, packaged and purified. A mice model of hepatic tumor will be built for gene therapy through ADI adenovirus. In order to discover the medicine security of ADI adenovirus, we will research its effects on cell physiology and pathology. ADI adenovirus' function on cell signal will be also studied deeply. We will try our best to reveal the molecular mechanism that ADI adenovirus induces cell apoptosis. The success of this project will not only supply a sample for targeting therapy of tumor, but also push the development of cancer gene therapy.

肿瘤基因治疗的靶向性和安全性问题一直都没有得到完全解决。我们研究发现，hTERT只在肿瘤细胞中表达，在正常细胞中沉默，表明hTERT的启动子对肿瘤细胞具有很好的靶向性；我们还发现，ADI的细胞内表达能高效杀死肝癌细胞，对正常肝细胞无影响。因此，课题组将利用Invitrogen公司的pAd/PL-DESTTM腺病毒载体，构建肿瘤靶向性的Promotor(hTERT)-ADI腺病毒和Promotor(TetO2)-ADI腺病毒。并将构建小鼠肝脏肿瘤的动物模型，实现ADI腺病毒的基因治疗，检测其治疗效果。然后研究ADI腺病毒对细胞和动物的生理、病理和毒理的影响，以及对肿瘤细胞信号通路的影响，深入揭示其诱导肝癌细胞凋亡的分子机制，探讨其对肿瘤基因治疗的安全性。此项研究的成功，不仅将对恶性肿瘤的靶向性治疗提供借鉴，还将有效推动肿瘤基因治疗的发展。

许多肿瘤在生长的过程中，因自身血管生长的不完善和缺陷，导致营养物质不能及时运送到肿瘤组织的所有部位，造成部分肿瘤组织始终处于饥饿状态，因此在临床上会有部分早期癌症患者体内的肿瘤一直不见生长，潜伏多年后转移爆发的现象。而且，在肿瘤的抗血管生成的靶向治疗过程中，肿瘤血管新生的抑制直接导致了肿瘤的营养饥饿。但单纯的抗血管生成的靶向治疗效果并不理想，存在极高的转移爆发的现象。一直以来，营养饥饿的肿瘤的生理病理机制和分子机制一直都不清楚。同时，肿瘤的饥饿疗法一直以来是毒副作用最小的辅助疗法，已经广泛应用于临床治疗。但是，由于饥饿治疗存在效率低、肿瘤靶向性差等劣势，因此饥饿治疗仅限于作为一种辅助治疗方式，需要进行进一步的改进。. 我们的研究工作主要分为四个层次。首先，为了提高肿瘤饥饿治疗的效率和靶向性，我们构建了端粒酶启动子hTERT调控表达的精氨酸脱亚氨基酶（Arginine deiminase, ADI）的腺病毒，用于肿瘤的精氨酸饥饿的基因治疗。该病毒在48小时内能造成肿瘤细胞约60%的凋亡率，正常细胞仅为30%左右，且对小鼠肿瘤模型中的肿瘤的溶瘤活性在6天内达到80%以上，具有巨大应用价值。其次，为了揭示精氨酸饥饿促进肿瘤细胞凋亡的分子机制，我们基于转录组的数据，对饥饿后的肿瘤细胞生理进行了深入的解析，揭示了精氨酸饥饿所造成的肿瘤细胞生理功能紊乱的分子机制。然后，为了解析饥饿促进肿瘤细胞扩散转移的分子机制，我们通过葡萄糖饥饿和精氨酸饥饿，揭示出ITGB8的上调导致RhoGTPase活性释放，从而促进细胞内肌动、肌球蛋白上调，导致细胞运动迁移能力上调的分子机制。最后，为了深入揭示精氨酸饥饿驱动肿瘤细胞凋亡的分子机制，我们通过代谢组的数据发现，精氨酸的饥饿能快速导致肿瘤细胞线粒体的损伤，从而造成肿瘤细胞快速凋亡。. 本项目的完成，一方面构建了能高效靶向性杀死肿瘤细胞的ADI腺病毒，具有潜在的临床应用价值；一方面精氨酸饥饿促进肿瘤细胞凋亡、转移的分子机制的揭示，有利于临床治疗癌症时，采用正确的应对机制，避免癌症的扩散和复发。