使用P19胚胎癌细胞的神经发生
1Department of Experimental Embryology, Institute of Genetics and Animal Breeding,Polish Academy of Sciences, 2Department of Regenerative Medicine,Maria Skłodowska-Curie Institute – Oncology Center, 3Department of Genomics and Biodiversities, Institute of Genetics and Animal Breeding,Polish Academy of Sciences
Summary
P19小鼠胚胎癌细胞系(P19细胞系)与体内分析相比,广泛用于神经发生分子机制的研究,具有极大的简化性。在这里,我们提出了P19细胞系中视黄酸引起的神经发生方案。
Abstract
从小鼠胚胎衍生的畸虫瘤衍生的P19细胞系具有分化成三个生殖层的能力。在视黄酸(RA)的存在下,悬浮培养的P19细胞系被诱导分化成神经元。这种现象作为体外神经发生模型被广泛研究。因此,P19细胞系对于与神经发生相关的分子和细胞研究非常有用。然而,文献中描述的P19细胞系神经元分化方案非常复杂。本研究开发的方法简单,对阐明神经发育异常和神经退行性疾病的分子机制起到一定的作用。
Introduction
在胚胎发育过程中,单个细胞层被转化为三个独立的生殖层1,2,3。为了增加在体内发生的现象的研究可能性,已经开发出三维聚集物(胚胎体)作为一种方便的模型。以这种方式形成的细胞聚集体可以暴露在各种导致细胞分化的条件下,这反映了胚胎4、5的发育。P19鼠胚胎癌细胞系(P19细胞系)常用作体外神经发生研究的细胞模型6、7、8。P19细胞系具有典型的多能干细胞特征,在细胞聚集过程中,在视黄酸(RA)存在的情况下可以分化成神经元,随后在粘附条件下生长出神经质。此外,未分化的P19细胞系还能够在二甲基硫酸盐(DMSO)9、10、11、12的影响下形成肌肉和心肌细胞样细胞。
许多方法13,14,15,16已经报告神经元分化,但方法有时很复杂,不容易掌握,只有阅读描述。例如,协议有时需要结合Dulbeco的改性鹰介质(DMEM)介质,辅以小牛血清(CS)和胎儿牛血清(FBS)13的混合物。此外,用于神经元发育的介质通常由神经巴沙和B27补充剂13,14,15,16组成。因此,现有方法在准备时包含复杂性,我们在此的目标是简化协议。在这项研究中,我们证明了使用FBS的DMEM可用于维持P19细胞系(DMEM = 10%FBS)以及神经元发育(DMEM = 5%FBS + RA)。这种使用P19细胞系的神经发生简化方法使我们能够研究神经元如何发育的分子机制。此外,对神经退行性疾病(如阿尔茨海默氏病)的研究也使用P19细胞系17,18进行,我们相信,本研究中开发的方法将起到阐明神经发育异常和神经退行性疾病的分子机制。
Protocol
1. 文化维护 培养维护介质中的P19细胞系(Dulbeco的改性Eagle培养基,含有4,500毫克/升葡萄糖,辅以10S、100单位/mL青霉素和100单位/mL链霉素)。在37°C和5%CO2下孵育。 2. 亚培养细胞 当细胞达到约80%汇合时,从细胞培养瓶中取出废培养基(表面积25cm2)。 用不含钙和镁的2 mL磷酸盐缓冲盐水(PBS)清洗细胞。 在细胞单层上加入1 mL的0.25%胰蛋白?…
Representative Results
图1给出了P19细胞系神经发生诱导方案的简化方案。为了在未分化状态和神经发生期间定义P19细胞系的特征,采用了RT-PCR(逆转录-聚合酶链反应)方法。未分化的P19细胞系表示多能基因,如有机阳离子/肉碱转运体4(Oct4)和Nanog主源盒(Nanog)。在RA存在的情况下,悬浮培养细胞聚集引起的神经发生导致Oct4和Nanog表达的迅速减少。相反,神?…
Discussion
在这里,我们描述了使用P19细胞系的神经发生的简单方案。虽然在这方面已经发表了许多报告,但使用P19细胞系进行神经发生诱导的详细方法仍不清楚。此外,我们在整个实验中使用了简单的高葡萄糖(4,500mg/L)DMEM培养基,10%FBS。这使我们能够以用户友好的方式进行神经生成实验,并在未来扩展此方法的使用范围。
该协议中最重要的点是RA浓度以及在悬浮培养液中生成细胞聚集。P19细?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了波兰国家科学中心(赠款No.UMO-2017/25/N/NZ3/01886)和KNOW(领先的国家研究中心)科学联盟”健康动物-安全食品”,科学和高等教育部第05-1/KNOW2/2015
Materials
| 6x DNA Loading Dye | EURx | E0260-01 | |
| Agarose | Sigma- Aldrich | A9539 | |
| cDNA synthesis kit | EURx | E0801-02 | |
| DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride) | Sigma- Aldrich | 10236276001 | Working concentration: 1 μg/mL |
| DMEM high glucose (4.5 g/L) with L-glutamine | Lonza | BE12-604Q | |
| Ethanol 99.8% | Chempur | CHEM*613964202 | |
| Fetal Bovine Serum (FBS) | EURx | E5050-03 | |
| MAP2 antibody | Thermo Fisher Scientific | PA517646 | Dilution 1:100 |
| PCR reaction kit | EURx | E0411-03 | |
| Penicillin/Streptomycin 10K/10K | Lonza | DE17-602E | |
| Phosphate Buffered Saline (PBS), 1x concentrated without Ca2+, Mg2+ | Lonza | BE17- 517Q | |
| Retinoic acid | Sigma- Aldrich | R2625-50MG | dissolved in 99.8% ethanol; store in -20 °C up to 6 months |
| Secondary Antibody (Alexa Fluor 488) | Thermo Fisher Scientific | A11034 | Dilution 1:500 |
| Skim milk | Sigma- Aldrich | 1153630500 | |
| TBE Buffer | Thermo Fisher Scientific | B52 | |
| Triton-X 100 | Sigma- Aldrich | T8787-100ML | |
| Trypsin 0.25% – EDTA in HBSS, without Ca2+, Mg2+,with Phenol Red | biosera | LM-T1720/500 | |
| Cell Culture Plastics | |||
| 1 mL Serological Pipettes | Profilab | 515.01 | |
| 10 mL Serological Pipettes | Profilab | 515.10 | |
| 100 mm dish dedicated for suspension culture | Corning | C351029 | |
| 15 mL centrifuge tubes | Sigma- Aldrich | CLS430791-500EA | |
| 5 mL Serological Pipettes | Profilab | 515.05 | |
| 6-well plate | Corning | CLS3516 | |
| Cell culture flasks, surface area 25 cm2 | Sigma- Aldrich | CLS430639-200EA |
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Cite This Article
Leszczyński, P., Śmiech, M., Teeli, A. S., Zołocińska, A., Słysz, A., Pojda, Z., Pierzchała, M., Taniguchi, H. Neurogenesis Using P19 Embryonal Carcinoma Cells. J. Vis. Exp. (146), e58225, doi:10.3791/58225 (2019).