星形胶质细胞来源细胞外囊泡促进大鼠神经干细胞向神经元的分化

Astrocyte-derived extracellular vesicles promote the differentiation of rat neural stem cells into neurons

背景:神经干细胞具有向神经元分化的潜能,已有报道表示星形胶质细胞与神经元存在密切联系,而有关星形胶质细胞来源细胞外囊泡是否具有促进神经干细胞向神经元高效分化的作用,仍未见相关报道。目的:探索星形胶质细胞来源细胞外囊泡诱导促进神经干细胞向神经元分化的可能性。方法:(1)体外培养大鼠神经干细胞及星形胶质细胞,行细胞形态及免疫荧光鉴定,进一步提取星形胶质细胞来源细胞外囊泡,并通过透射电镜、粒径分析、Western blot等方法进行鉴定;(2)将第3代神经干细胞分2组,分别加入同体积的PBS、星形胶质细胞来源细胞外囊泡(质量浓度为1 mg/mL)干预6 d;(3)通过免疫荧光、qRT-PCR和Western blot检测星形胶质细胞来源细胞外囊泡干预后神经元标志物的表达水平。结果与结论:(1)显微镜下神经干细胞形态为球形,以细胞团状分布,免疫荧光示特征性标志物CD133、Nestin呈高表达水平;(2)显微镜下星形胶质细胞形态为不规则星状,且免疫荧光示特征性标志物胶质纤维酸性蛋白呈高表达水平;(3)星形胶质细胞来源细胞外囊泡形态近似圆形或椭圆形杯状,动态光粒子散射仪显示直径在10-200 nm之间,Western blot示细胞外囊泡相关标志物CD63及TSG101表达阳性;(4)相比PBS组,星形胶质细胞来源细胞外囊泡组可见神经干细胞大量贴壁分化,分化细胞胞体饱满,有明显突起,主要呈神经元样,且微管蛋白β3及神经丝蛋白200呈高表达(P<0.05);(5)以上结果提示,星形胶质细胞来源细胞外囊泡可促进神经干细胞向神经元分化。

BACKGROUND:Neural stem cells have the potential to differentiate into neurons.It has been reported that astrocytes are closely related to neurons,but whether astrocyte-derived extracellular vesicles can promote the efficient differentiation of neural stem cells into neurons has not been reported.OBJECTIVE:To explore the possibility of inducing and promoting neuronal differentiation of neural stem cells from extracellular vesicles derived from astrocytes.METHODS:(1)Rat neural stem cells and astrocytes were cultured in vitro and identified by cell morphology and immunofluorescence.Astrocyte-derived extracellular vesicles were further extracted and identified by transmission electron microscopy,particle size analysis,western blot assay and other methods.(2)Passage 3 neural stem cells were divided into two groups:Neural stem cells were treated with the same volume of PBS and astrocyte-derived extracellular vesicles(1 mg/mL)for 6 days.(3)The expression levels of neuron markers after astrocyte-derived extracellular vesicle intervention were detected by immunofluorescence,qRT-PCR and western blot assay.RESULTS AND CONCLUSION:(1)Under the microscope,neural stem cells were spherical in shape and distributed in cell clusters.Immunofluorescence showed that the characteristic markers CD133 and Nestin were highly expressed.(2)Astrocyte morphology was irregular stellate under the microscope,and immunofluorescence showed that the characteristic marker glial fibrillary acidic protein was highly expressed.(3)The shape of astrocyte-derived extracellular vesicles was round or oval cup and 10-200 nm in diameter by dynamic light particle scatterer.Western blot assay showed positive expression of CD63 and TSG101,related markers of extracellular vesicles.(4)Compared with PBS group,astrocyte-derived extracellular vesicle group showed a large number of adherent differentiation of neural stem cells,and the differentiated cells were plump and obvious protrusions,which were mainly neuron-like,and the expression of tubulinβ3 and neurofilament 200 was high(P<0.05).(5)These results suggest that astrocyte-derived extracellular vesicles can promote the differentiation of neural stem cells into neurons.