神经干细胞与大鼠RPE细胞间的线粒体转运及对RPE细胞凋亡的影响

Mitochondrial transfer from neural stem cells rescues apoptosis of retinal pigment epithelial cells isolated from RCS rats

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 pattern of mitoehondrial transfer in neural stem cells （NSCs） and primary cultured retinal pigment epithelial （RPE） cells isolated from Royal College of Surgeons （RCS） rats and analyze the effect mitochondrial transfer on the biological behaviors of RPE cells. Methods The RPE cells were isolated from the RCS rats, cultured primarily and identified. Mitotracker-red and mitotracker-green staining were used to label the mitochondria of the cultured RPE cells and those of mouse neural stem cell line C17.2, respectively. In a co-culture system of the 2 kinds of cells, the formation of tunneling nanotubes （TNT） between them and mitochondrial transfer in the TNT were observed by laser scanning confocal microscopy. Flow cytometry was used to measure the level of reactive oxygen species （ROS） production and analyze the cell cycle distribution and early apoptosis of RPE cells cultured alone or in the co- culture system. Results The RPE cells in the third generation showed a good growth condition with the positive rates for RPE65 and Bestrophin both exceeding 95%. At 24 h after the co-culture, the formation of TNT structure was observed between RPE cells and NSCs, and one-way mitochondria] transfer from NSCs to RPE cells were captured. Mitochondrial transfer from the NSCs to the RPE cells resulted in significantly decreased ROS level in the latter cells （ P 〈 0.05 ）. The RPE cells receiving the mitochondria also showed enhanced cell proliferation, increased cell proportion in S phase （ P 〈 0. 01 ） , elevated proliferation index （P 〈 0. 05）, and decreased early apoptosis cells （P 〈 0. 05 ）. Conclusion In the co-culture system, NSCs transfer mitochondria into adjacent RPE cells via the TNT structure to rescues the RPE cells from apoptosis.