应用Nissl/GFAP荧光标记流式细胞术检测神经干细胞分化的研究

Study on the differentiation of neural stem cells by flow cytometry with Nissl/GFAP fluorescent labeling

目的:探寻一种能快速准确地对神经干细胞分化结果进行定量鉴定的检测方法。方法:采用神经元核抗原(neuronal nuclei,NeuN)免疫荧光和Nissl荧光染色观察体内神经元尼氏小体的染色情况;采用NeuN免疫荧光和Nissl荧光染色观察体外神经干细胞诱导分化后的神经元尼氏小体的荧光染色情况;体外诱导神经干细胞分化后,收集细胞悬液进行胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫荧光和Nissl荧光标记,显微镜和流式细胞术检测神经干细胞分化情况。结果:NeuroTrace Nissl 530/615红色荧光染色剂除了对体内、外NeuN阳性神经元的尼氏小体染色外,所有细胞的细胞核也呈阳性信号;神经干细胞分化后的细胞悬液滴片可观察到GFAP免疫荧光阳性的星形胶质细胞,其Nissl荧光仅能标记细胞核,而GFAP阴性细胞除细胞核阳性信号外,还可观察到尼氏小体;流式细胞术可通过GFAP和Nissl两个荧光通道明显区分神经干细胞诱导分化后的神经元和胶质细胞。结论:应用Nissl/GFAP荧光标记流式细胞术可快速准确地计数神经干细胞诱导分化后的神经元和胶质细胞的数量及比例。

Objective:To explore a rapid and accurate method for quantitative identification of neural stem cell differentiation.Methods:Neuronal nuclei(NeuN)immunofluorescence and Nissl fluorescente staining were used to observe the staining of Nissl bodies of neurons in vivo;NeuN immunofluorescence and Nissl fluorescence staining were used to observe the fluorescence staining of Nissl bodies after differentiation of neural stem cells in vitro.After inducing neural stem cells to differentiate in vitro,the cell suspension was collected for glial fibrillary acidic protein(GFAP)immunofluorescence and Nissl fluorescence staining,and the differentiation of neural stem cells was detected by microscope and flow cytometry.Results:NeuroTrance Nissl 530/615 dark red fluorescent Nissl staining not only stained Nissl bodies of NeuN positive neurons in vivo and in vitro,but also showed positive signals in the nuclei of all cells.The Nissl fluorescence staining of GFAP positive astrocytes could only label the nucleus,while the Nissl bodies of GFAP negative cells could be observed in addition to the positive signal of nucleus.GFAP and Nissl fluorescence channels can be used to differentiate neurons and glial cells induced by neural stem cells by flow cytometry.Conclusion:Nissl/GFAP flow cytometry can quickly and accurately count the number and proportion of neurons and glial cells after neural stem cells differentiation.