SH-SY5Y 细胞分化后表型标志物的定量特征分析。

Quantitative Characterization of Phenotypical Markers After Differentiation of SH-SY5Y Cells.

机构信息

Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, 3001 Bern, Switzerland.

Department of Neurosurgery, Research Unit, Inselspital, University of Bern, 3010 Bern, Switzerland.

出版信息

CNS Neurol Disord Drug Targets. 2020;19(8):618-629. doi: 10.2174/1871527319666200708132716.

DOI:10.2174/1871527319666200708132716

PMID:32640966

Abstract

BACKGROUND

The human neuroblastoma cell line, SH-SY5Y, has been widely used in neuroscience research, especially in studies related to Parkinson's disease. However, differences between clones have been demonstrated, highlighting the importance to characterize the properties of this cell line carefully.

OBJECTIVE

The aim of this study was to characterize the phenotype of undifferentiated and differentiated SH-SY5Y cells using various differentiation protocols.

METHODS

A morphological and quantitative analysis of markers related to dopaminergic and cholinergic neurons, but also other phenotypes, was performed.

RESULTS

Differentiated cells showed the typical neuronal morphology. Undifferentiated cells expressed low levels of Tyrosine Hydroxylase (TH) and higher levels of the high-affinity Choline Transporter (CHT1). Staurosporine (ST)-differentiation resulted in the highest number of THimmunoreactive cells, followed by phorbol ester Phorbol-12-Myristate-13-Acetate (PMA), whereas differentiation with Brain-Derived Neurotrophic Factor (BDNF) did not increase TH-immunoreactive cells. TH, dopamine β-hydroxylase and vesicular monoamine transporter-2 were also significantly upregulated in ST-differentiated cells compared to both undifferentiated and Retinoic Acid (RA)- differentiated cells. RA induced the highest number of CHT1-immunoreactive cells while ST- and BDNF-differentiation reduced CHT1-immunoreactive cells, indicating a decrease in the cholinergic phenotype. The presynaptic neuronal protein, α-synuclein, was significantly upregulated in RA- and ST-treated cells compared to undifferentiated cells. Ascorbic acid increased the number of CHT1-immunoreactive cells in all differentiation procedures and ST-differentiated TH-positive cells significantly.

CONCLUSION

Our findings indicate that a quantitative characterization of the phenotype is crucial when using SH-SY5Y cells to study the pathogenesis or evaluate compounds for treatment of neurodegenerative diseases.

摘要

背景

人神经母细胞瘤细胞系 SH-SY5Y 广泛应用于神经科学研究，特别是帕金森病相关研究。然而，已有研究证实不同克隆株之间存在差异，这突出表明需要仔细描述该细胞系的特性。

目的

本研究旨在通过多种分化方案，对未分化和分化的 SH-SY5Y 细胞的表型进行特征描述。

方法

对与多巴胺能和胆碱能神经元相关的标志物，以及其他表型进行形态学和定量分析。

结果

分化后的细胞呈现典型的神经元形态。未分化细胞表达低水平的酪氨酸羟化酶（TH）和高水平的高亲和力胆碱转运体（CHT1）。用星形孢菌素（ST）诱导分化产生的 TH 免疫反应性细胞数量最多，其次是佛波醇酯佛波醇-12-肉豆蔻酸-13-乙酸酯（PMA），而用脑源性神经营养因子（BDNF）诱导分化则不会增加 TH 免疫反应性细胞。与未分化细胞和维甲酸（RA）分化的细胞相比，ST 分化的细胞中 TH、多巴胺 β-羟化酶和囊泡单胺转运体-2 的表达也显著上调。RA 诱导产生的 CHT1 免疫反应性细胞数量最多，而 ST 和 BDNF 分化则减少 CHT1 免疫反应性细胞，表明胆碱能表型减少。与未分化细胞相比，RA 和 ST 处理的细胞中突触前神经元蛋白α-突触核蛋白的表达显著上调。抗坏血酸（ascorbic acid）增加了所有分化程序中的 CHT1 免疫反应性细胞数量，且显著增加了 ST 分化的 TH 阳性细胞数量。