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目的研究小鼠神经干细胞(neural stem cells,NSCs)C17.2细胞系与从RCS大鼠变性视网膜中原代分离视网膜色素上皮细胞(retinal pigment epithelial,RPE)的线粒体交换方式及其对RPE细胞特性的影响。方法分离RCS大鼠RPE细胞并进行培养和鉴定。分别用线粒体特异性标记物Mitotracker-red和Mitotracker-green标记RPE细胞和小鼠NSCs细胞的线粒体,将两种细胞共培养,激光共聚焦显微镜下观察两种细胞之间隧道纳米管(tunneling nanotubes,TNT)的形成及线粒体转运方式。采用流式细胞仪检测与NSCs共培养后RPE细胞的反应性活性氧类(reactive oxygen species,ROS)水平、细胞周期和细胞凋亡水平的变化。结果来源于RCS大鼠视网膜的第3代RPE细胞生长状态良好,RPE65及Bestrophin蛋白阳性率细胞均大于95%。与NSCs共培养24 h后,可见RPE细胞与NSCs间TNT的形成,NSCs中的线粒体向RPE细胞方向单向运动,接受NSCs转运的线粒体后,RPE细胞的ROS水平降低(P<0.01);RPE细胞的增殖能力增加,处于S期的细胞比例明显增加(P<0.01),细胞增殖指数(PI)显著增加(P<0.05);RPE细胞早期凋亡细胞比例显著降低(P<0.05)。结论 NSCs可通过与相邻的变性RPE细胞之间形成TNT并向其转运线粒体而改善变性RPE细胞的存活。
Objective To investigate the mitochondrial exchange pattern of mouse primary neural stem cells (NSCs) C17.2 cell line and primary retinal pigment epithelial (RPE) cells from the degeneration retina of RCS rats and their effects on the characteristics of RPE cells influences. Methods RPE cells from RCS rats were isolated, cultured and identified. Mitochondria of RPE cells and mouse NSCs were labeled with mitochondria-specific markers Mitotracker-red and Mitotracker-green respectively. The two kinds of cells were co-cultured, and under the confocal laser scanning microscope, the tunneling nanotubes TNT) formation and mitochondrial transport mode. The changes of reactive oxygen species (ROS), cell cycle and apoptosis in RPE cells after co-culture with NSCs were detected by flow cytometry. Results The third generation of RPE cells derived from the retina of RCS rats grew well, and the positive cells of RPE65 and Bestrophin protein were more than 95%. After co-cultured with NSCs for 24 h, the formation of TNT between RPE cells and NSCs was observed. The mitochondria in NSCs migrated to RPE cells in a unidirectional manner. After transfected with mitochondria, the ROS levels in RPE cells decreased (P <0.01) (P <0.01). The proliferation index (PI) increased significantly (P <0.05). The proportion of early apoptotic cells in RPE cells was significantly decreased (P <0.05). Conclusion NSCs can improve the survival of degenerative RPE cells by forming TNT with adjacent denatured RPE cells and transporting them to mitochondria.