线粒体移植增强人神经胶质瘤U87细胞的辐射敏感性

线粒体移植( mitochondrial transplantation)是从患者正常组织分离线粒体然后注入线粒体损伤或缺失的部位，使损伤细胞获得救治、器官功能得以恢复的全新干预技术。本研究重点探讨线粒体移植对人神经胶质瘤细胞(U87)辐射敏感性的影响。提取人星形胶质细胞(human astrocytes, HA)的线粒体，用荧光探针Mito-Tracker Red进行标记后再与U87细胞共培养，激光共聚焦显微镜观察发现，Mito-Tracker Red的红色荧光大量出现在U87细胞内；随后对进入细胞内的游离线粒体荧光强度进行定量分析，游离线粒体和U87细胞共培养12 h时，单细胞内荧光强度由本底值0.08上升至1.83，表明游离线粒体可以通过共培养方式进入U87细胞内。随后给予U87细胞X射线辐照，Western印迹结果显示，联合组与单独辐照组相比，Cyto-C和Bax的表达量分别由179.5%和198.5%升高至251.72%和256.10%，Bcl-2的表达量由57.17%降低至22.23%；使用Annexin V/PI双染法检测到联合组U87细胞凋亡量相比单独辐照组的3.1%和23.5%上升至19.2%和43.8%；RT-CES检测结果表明，96 h内联合组U87细胞生长曲线被抑制；联合组克隆形成率相对单独辐照组的53%下降至29.3%。鬼笔环肽染色发现，线粒体移植组细胞表面丝状伪足相比对照组明显减少。本研究提示，线粒体移植能够促进辐射诱导的肿瘤细胞凋亡，对U87细胞具有辐射增敏作用，其作用机制与重新激活线粒体凋亡通路、降低肿瘤恶性程度有关。

Mitochondrial Transplantation Enhances Radiosensitivity of Human Glioma U87 Cells

Mitochondrial transplantation, is a novel therapeutic intervention by isolating the mitochondria from the normal parts of the patient and then injecting into the areas in which mitochondria are deficient, allowing the injured cells and organs return to normal function. This study focused on the effects of mitochondrial transplantation on radiosensitivity of human glioma (U87) cells. Isolated mitochondria from human astrocytes (HA) cells were extracted and labeled with Mito-Tracker Red and then co-cultured with U87 cells. The red fluorescence of Mito-Tracker Red was observed in the U87 cells by laser confocal microscopy. Quantitative analysis showed that the intracellular fluorescence intensity of free mitochondria and U87 cells increased from 0.08 to 1.83 after co-culture for 12 h, indicating that isolated mitochondria could enter U87 cells by co-culture. U87 cells were then exposed to X-rays. Western blot results showed that the expression levels of Cyto-C and Bax were increased from 179.5% and 198.5% to 251.72% and 256.10%. The expression of Bcl-2 was decreased from 57.17% to 22.23% in the combined group compared with the single irradiation group. The apoptosis of U87 cells in the combined group was detected by Annexin V/PI double staining, which increased to 19.2% and 43.8% as compared with 3.1% and 23.5% of the irradiated group alone. The results of RT-CES showed that the growth curve of U87 cells was inhibited in 96 h. The colony-forming ratio of combined group decreased from 53% to 29.3% when compared with that of irradiated group. In addition, after staining with phalloidin, the number of filopodia in the mitochondrial transplantation group was obviously reduced. Overall, these findings suggested that mitochondrial transplantation could enhance the radiosensitivity of U87 cells, which may be related to the activation of mitochondrial apoptotic pathway after X-ray radiation and the reduction of malignancy of the tumor cells.