TERT线粒体转移介导骨肉瘤干细胞耐药及其机制研究

The drug resistance is a great challenge in clinical setting, cancer stem cell is thought to closely involved. Previous study has showed that telomerase positive osteosarcoma cells possessed stem cell-like properties and were resistant to chemotherapeutic reagents, however, the underlying mechanism is unclear. It is reported that TERT also existed in mitochondrion, and TERT relocalized to mitochondrion in response to oxidative stress. In this study, we assume that TERT induces chemoresistance through translocating to mitochondrion and inhibiting mitochondrial apoptosis pathway. To examine the above hypothesis, we first study the TERT expression and localization in cisplatin/doxorubicin-resistant osteosarcoma cells using immunoblot, immunofluorescence and Immune colloidal gold, so as to observe whether TERT translacates to mitochondria in response to chemotherapeutic stress. On this basis, we then construct inducible TERT express/interference lentivirus. Osteosarcoma cells are infected and selected for stably transfected cells. To determine the relationship between the TERT and osteosarcoma cells in vitro and in vivo, the cells or xenograft are divided into different groups according to their TERT expression status. Furthermore, we will determine the effect of TERT on the mitochondria apoptosis pathway under chemotherapeutic stress and investigate the underlying mechanism. The present study aims at elucidating the relationship between TERT mitochondria translocation and chemo-resistance of osteosarcoma stem cells, and provide the basis for cancer stem cell targeted therapy in osteosarcoma.

骨肉瘤的耐药是临床上亟待解决的难题，肿瘤干细胞与之密切相关。前期研究表明端粒酶阳性骨肉瘤细胞具有干细胞样特性，且其对化疗耐药，但其具体机制不明。研究发现TERT也存在于线粒体中，在氧化应激时存在TERT向线粒体重定位现象。本研究推测TERT通过定位至线粒体并抑制线粒体凋亡途径介导耐药，为证实上述假设，首先利用免疫印迹、免疫荧光及免疫胶体金法检测化疗耐药细胞中TERT的表达量及定位，观察TERT在耐药情况下是否向线粒体转移。在此基础上，构建TERT诱导型过表达及干扰慢病毒，感染并获得稳转细胞，通过上调或下调TERT探讨其对骨肉瘤化疗敏感性的影响，进一步检测TERT变化对线粒体凋亡途径的影响，探讨其介导耐药的机制。本项目旨在探讨TERT线粒体转移与骨肉瘤化疗耐药的关系，为骨肉瘤干细胞靶向治疗提供理论基础。

骨肉瘤的耐药是临床上亟待解决的难题，肿瘤干细胞与之密切相关。本项目首先利用免疫荧光及免疫印迹法检测顺铂/阿霉素耐药细胞中TERT的表达量及定位，证实骨肉瘤耐药细胞中TERT表达量明显升高，且骨肉瘤耐药细胞中出现TERT线粒体转移。其次，成功构建TERT过表达及干扰慢病毒，实现TERT上调或下调，结果表明TERT能够抑制顺铂/阿霉素诱导的凋亡，且这种效应与端粒酶活性无关。机制方面，利用免疫印迹技术，表明TERT通过增加Bcl-2/Bax比例，作用于线粒体凋亡通路，减少顺铂/阿霉素介导的骨肉瘤细胞凋亡。通过流式细胞术，发现TERT通过减少细胞内活性氧（ROS）从而抑制顺铂/阿霉素诱导的骨肉瘤凋亡。利用小鼠移植瘤实验，体内证实TERT能够增强骨肉瘤细胞对顺铂的耐药性，且联合端粒酶抑制剂及Bcl2 抑制剂能够协同抑制骨肉瘤。这些研究表明TERT 线粒体转移参与骨肉瘤干细胞化疗耐药，靶向骨肉瘤TERT线粒体转移能够增强其化疗敏感性，为骨肉瘤干细胞靶向治疗提供理论基础。