肌萎缩侧索硬化患者脑脊液对U251细胞隧道纳米管形成的影响

Effects of cerebrospinal fluid from patients with amyotrophic lateral sclerosis on generation of tunneling nanotubes-like structure in U251 cells

目的:探讨肌萎缩侧索硬化(ALS)患者脑脊液对神经胶质细胞隧道纳米管(TNTs)形成的影响。方法:收集16例ALS患者的脑脊液(ALS-CSF)及10例功能性头痛患者的脑脊液(对照),分别用于培养U251细胞0、3、7、10、14 d,采用免疫荧光显微镜观察TNTs的形成,记录表达TNTs结构的U251细胞百分比,采用Western blot法检测ALS-CSF培养0、3、7、10、14 d后U251细胞中F-actin蛋白的表达。结果:在ALS-CSF培养条件下,表达TNTs结构的U251细胞百分比较对照组增加(P<0.05),且表达TNTs结构的U251细胞百分比在3 d时开始增加,7 d时达到高峰,10 d时开始逐渐下降;ALS-CSF培养的U251细胞F-actin表达量在3 d时升高,在7 d时表达量最高,在10d时开始下降。结论:ALS-CSF培养的U251细胞在早期可诱导TNTs的形成,可能促使ALS相关致病蛋白通过TNTs在中枢神经系统中播散。

Aim： To investigate the effects of cerebrospinal fluid from patients with amyotrophic lateral sclerosis（ ALSCSF） on the generation of tunneling nanotubes（ TNTs）-like structure in the human glioma U251 cells. Methods： CSF was collected from 16 ALS patients and 10 persons with functional headache（ control） and used to culture U251 cells for 0,3,7,10,14 days,respectively. The generation of TNTs was observed under fluorescence microscope,and the percentage of U251 cells with TNTs among U251 cells was recorded. Then Western blot was used to detect the expression of F-actin in U251 cells cultured with ALS-CSF for 0,3,7,10,and 14 days. Results： The percentage of U251 cells with TNTs among U251 cells cultured with ALS-CSF was higher than control,which started to increase at day 3,and reached the peak at day 7,then gradually decreased from day 10. The expression of F-actin in U251 cells cultured with ALS-CSF had the same trend.Conclusion： ALS-CSF could induce the generation of TNTs-like structure in U251 cells,and the ALS-related pathogenic protein may propagate through TNTs-like structure in central nervous system of patients with ALS.