星形胶质细胞分泌的外泌体对神经干细胞活力的影响研究

The Effect of Exosomes Secreted by Astrocytes on the Vitality of Neural Stem Cells

目的探讨小鼠星形胶质细胞外泌体对神经干细胞活力的影响。方法分离培养小鼠星形胶质细胞,收集细胞上清后超离出外泌体并予以鉴定。取原代培养的第2~6代神经干细胞,分别以0、20、40、60μg/mL外泌体的条件培养基处理,CCK-8法筛选出培养神经干细胞的最佳外泌体质量浓度(40μg/mL)。实验分为实验组(40μg/mL外泌体处理的神经干细胞)和对照组(加入同等体积PBS处理的神经干细胞),干预72 h后,EdU试剂盒标记阳性干细胞数。利用Transwell模型,通过4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole, DAPI)染色,荧光显微镜下分别统计出Transwell下室的实验组和对照组的细胞核个数。结果①星形胶质细胞外泌体的鉴定:通过电镜、蛋白免疫印迹实验、外泌体浓度粒径等技术鉴定细胞上清超离出的外泌体;②CCK8实验检测:随着外泌体质量浓度的增加,促进原代神经干细胞的增殖作用逐渐增强,且与对照组相比,40μg/mL、60μg/mL外泌体组对神经干细胞均有显著增殖作用,但此两组间比较无明显差异,所以选择40μg/mL为最佳干预质量浓度;③EdU检测:实验组EdU标记阳性细胞数高于对照组(P<0.05);④Transwell模型中,实验组处理的神经干细胞从Transwell膜上层迁移到下层的细胞个数高于对照组(P<0.05)。结论小鼠星形胶质细胞外泌体可提高神经干细胞的生存活力。

Objective To investigate the effects of exosomes of mouse astrocytes on the viability of neural stem cells. Methods Cultured and isolated the mouse astrocytes, and collected the cell supernatant for obtain the exosomes by ultracentrifugation. Neural stem cells that primary cultured for 2 nd to 6 th generation were obtained and treated with medium contained 0, 20, 40, 60 μg/mL of exosomes respectively. Screening the optimal exosome concentration for culturing neural stem cells by CCK-8 method. The optimal exosome concentration for neural stem cells was 40 μg/mL according to CCK-8 results. Then cells were intervened with 40 μg/mL of exosome in experimental group for 72 h, and the control group was added with the same volume of PBS. After intervention, the positive stem cells were labeled with EdU kit. Using the Transwell model, the number of nucleus stained by DAPI in the lower chamber in 40 μg/mL exosome treatment group and the control group were counted under a fluorescence microscope. Results ① Identification of astrocyte exosomes: The successful obtain of exosomes of cell supernatant were confirmed by techniques such as electron microscopy, Western blot, exosome concentration and particle size measurement. ② CCK8 experiment: As the increasement of the concentration of exosomes, cell proliferation of primary neural stem cells gradually increased.Compared with the control group, proliferation of the cells in 40 μg/mL and 60 μg/mL exosome treatment groups was significantly enhanced, but there was no significant difference between the two groups. So, 40 μg/mL was selected as the best intervention concentration. ③ EdU detection: Number of EdU positive labeled cells in the 40 μg/mL exosome group was higher than that in the control group(P<0.05). ④ Transwell experiment: In the Transwell model, more neural stem cells in the 40 μg/mL exosome group migrated from the upper layer to the lower layer of the Transwell membrane, and the number was higher than that of the control group(P<0.05). Conclusion Mouse astrocyte exosomes can improve the viability of neural stem cells.