在SH-SY5Y人神经母细胞瘤分化
Summary
It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.
Abstract
Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease.
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.
Introduction
体外模型系统中使用的能力已大大增强神经生物学和神经科学领域。培养中的细胞提供了一个高效的平台来表征蛋白质的功能和分子机制的具体现象，了解疾病和感染的病理，并进行初步的药物测试评估。在神经生物学，主要类型的细胞培养模型包括例如大鼠B35细胞5，神经2A小鼠细胞6和大鼠PC12细胞7从大鼠和小鼠，以及成神经细胞瘤细 胞系的原代神经元培养物。虽然使用这样的细胞系的已显著先进领域，有与处理非人类细胞和组织相关的几个混杂因素。相比于人体时，这些包括理解特定物种的代谢过程的差异，疾病的表现，和发病机制的表型。同样重要的是要注意，再是小鼠和人的基因表达和转录因子信号之间显著差异，突出啮齿动物模型的局限性和谅解其途径是啮齿动物和人类8-11之间是保守的重要性。其他已经采用了利用人体的神经元细胞系，包括N型的Tera-2（NT2）人畸胎瘤细胞和诱导多能干细胞（iPS细胞）的。这些细胞系为体外人类系统不错的机型。然而，NT2细胞用视黄酸（RA）的结果的神经元，星形胶质细胞的混合群的产生，和径向胶质细胞12，因此需要另外的纯化步骤的分化得到的神经元的纯群体。此外，NT2细胞表现出高度可变的核型13，具有在细胞的72％大于60染色体。 iPS细胞表现出变异性在不同细胞系之间的差异，并在分化效率变化 14，它因此希望具有一个一致的和可重复的人神经元细胞模型，以补充这些替代。
SH-SY5Y成神经细胞样细胞是亲神经母细胞瘤细胞系SK-N-SH的亚克隆。从包含两个成神经细胞样细胞和上皮样细胞15骨髓活检于1970年产生的亲本细胞系。 SH-SY5Y细胞具有由47条染色体的稳定核型，并且可以从一个神经细胞样状态成成熟的人神经元，通过各种不同的机制，包括使用的RA，佛波酯和特定神经营养蛋白诸如脑源性分化神经营养因子（BDNF）。现有的证据表明，使用不同的方法，可以为特定的神经元亚型如肾上腺素，胆碱，和多巴胺能神经元16,17选择。这后一方面使得的神经生物学实验的大量有用的SH-SY5Y细胞。
内容】“>几项研究已经注意到在其未分化和分化的状态SH-SY5Y细胞之间的重要区别。当SH-SY5Y细胞是未分化的，它们迅速增殖并似乎非偏振光，除了极少数，短流程。它们通常生长表示未成熟的神经元18,19的中块和表达的标志物。当有区别的，这些细胞扩展长的，支链的过程，减少增殖，并且在一些情况下，极化2,18。完全分化的SH-SY5Y细胞先前已经证明表达多种成熟神经元包括生长相关蛋白（GAP-43），神经元核（的NeuN），突触素（SYN），突触小泡蛋白II（SV2），神经元特异性烯醇化酶（NSE）和微管相关蛋白（MAP）的不同的标记的2,16,17,20，和缺乏神经胶质标记物，如胶质纤维酸性蛋白（GFAP）4的表达。在进一步的支持，分化SH-SY5Y细胞represeNT均匀的神经元群，拆除BDNF的导致细胞凋亡4。这表明，分化SH-SY5Y细胞的存活依赖于营养因子，类似于成熟神经元。SH-SY5Y细胞的使用增加了，因为亚克隆成立于1978年3。它们的使用的一些实例包括：调查帕金森氏病17，阿尔茨海默氏病21，和病毒感染，包括脊髓灰质炎病毒22，肠道病毒71型的发病23,24 ，水痘带状疱疹病毒（VZV）1，人巨细胞病毒25，和单纯疱疹病毒（HSV）2,26。使用SH-SY5Y细胞在他们的未分化的形式使用这些细胞要注意，一些研究，特别是在neurovirology 27-36领域是很重要的。在的未分化与分化的SH-SY5Y细胞中观察到的表型的差异引起的磨片的问题疗法感染的观察进展会在成熟分化的神经元的不同。例如，分化的SH-SY5Y细胞具有HSV-1的摄取与未分化，增殖SH-SY5Y细胞，这可能是因为缺乏结合HSV并调节上未分化的SH-SY5Y细胞2条目表面受体的一个更高的效率。因此，关键是设计侧重于测试在体外的神经元的实验时，SH-SY5Y细胞应以获得翻译和对比的最准确的结果，以在体内模型区分。
一个可靠的方法的发展，产生人类神经细胞当务之急是让研究人员进行翻译实验证明，精确地模拟人的神经系统。这里介绍的协议是界定从以前的方法得出1-4最佳实践来丰富对于那些分化的人神经过程使用视黄酸。
Protocol
Representative Results
Discussion
上述协议提供了一个直接的和可重复的方法来生成均匀的和可行的人类神经文化。该协议利用技术和集成多个先前公布的方法1-4和目标划定每个最佳实践做法。 SH-SY5Y细胞的分化依赖于逐渐血清剥夺;加入视黄酸，神经营养因子和细胞外基质蛋白;和串行分裂来选择分化成熟的神经细胞贴壁。该细胞系开始以贴壁和悬浮的细胞的异质群体。该协议的目的是通过传代或媒体变更前预提PBS洗涤以?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful for the contributions of Yolanda Tafuri in optimizing conditions for SH-SY5Y differentiation, and for the support of Dr. Lynn Enquist, in whose lab this work was initiated. Y. Tafuri contributed the images shown in Figure 3. This work was supported by the NIH-NIAID Virus Pathogens Resource (ViPR) Bioinformatics Resource Center (MLS and L. Enquist) and K22 AI095384 (MLS).
Materials
| B-27 | Invitrogen | 17504-044 | See Table 1 for preparation |
| Brain-Derived Neurotrophic Factor (BDNF) | Sigma | SRP3014 (10ug)/B3795 (5ug) | See Table 1 for preparation |
| dibutyryl cyclic AMP (db-cAMP) | Sigma | D0627 | See Table 1 for preparation |
| DMSO | ATCC | 4-X | – |
| Minimum Essential Medium Eagle (EMEM) | Sigma | M5650 | – |
| Fetal Bovine Serum (FBS) | Hyclone | SH30071.03 | See Table 1 for preparation |
| GlutamaxI | Life Technologies | 35050-061 | – |
| Glutamine | Hyclone | SH30034.01 | – |
| Potassium Chloride (KCl) | Fisher Scientific | BP366-1 | See Table 1 for preparation |
| MaxGel Extracellular Matrix (ECM) solution | Sigma | E0282 | See step 11 of the protocol |
| Neurobasal | Life Technologies | 21103-049 | – |
| Penicillin/Streptomycin (Pen/Strep) | Life Technologies | 15140-122 | – |
| Retinoic acid (RA) | Sigma | R2625 | Should be stored in the dark at 4° C because this reagent is light sensitive |
| SH-SY5Y Cells | ATCC | CRL-2266 | – |
| 0.5% Trypsin + EDTA | Life Technologies | 15400-054 | – |
| Falcon 35mm TC dishes | Falcon (A Corning Brand) | 353001 | – |
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