小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化
Summary
本文介绍了培养小鼠髓质前体32维/G-脑脊液 R 细胞系的详细规程, 并进行了病毒感染, 并进行了增殖和分化测定。该细胞系适合研究髓细胞发育, 以及兴趣基因在髓细胞生长和中性分化中的作用。
Abstract
对造血干细胞和祖细胞生物学的理解对再生医学和血液病理学的治疗具有重要意义。尽管可以使用体内模型或初级区域性获取最相关的数据, 但造血干细胞和祖细胞的低丰度大大限制了适当技术的研究。因此, 使用细胞线可以充分生产生物材料, 以执行需要大量细胞数的筛查或化验。在这里, 我们提供了一个详细的描述, 读数, 和解释的增殖和分化的检测, 用于调查过程中涉及 myelopoiesis 和中性分化。这些实验采用32维/克-脑脊液-R 细胞因子依赖的小鼠髓细胞系, 具有在 IL-3 存在时增殖的能力, 并能分化为 g 脑脊液。我们为处理32维/克-脑脊液 R 细胞提供了最优化的协议, 并讨论了可能危及所描述的化验结果和预期效果的主要缺陷和缺点。此外, 本文还包含了慢病毒载体和逆转录病毒的生产、滴定和转导32维/克-脑脊液 R 细胞的协议。我们证明, 这些细胞的遗传操作可以用来成功地进行功能和分子研究, 这可以补充取得的结果与原发性造血干细胞和祖细胞或在体内模型。
Introduction
造血干细胞和祖细胞为生物体提供了大量成熟的细胞, 包括来自髓系谱系 (中性粒、嗜酸性细胞、嗜碱性细胞和单核) 的干细胞。推动从造血干细胞生产髓细胞的过程被称为 myelopoiesis, 适当地生产成熟的髓细胞以响应不断变化的需求是适当应对压力的有机体的先决条件。条件, 如感染和失血。成熟髓细胞的生产不足可能导致无法消除病原体, 减少凝血和其他危及生命的条件1,2。此外, 髓系发育的改变也可能与血液恶性肿瘤有关, 如急性髓细胞白血病 (AML)3。myelopoiesis 的改变可能是由于各种原因造成的, 例如细胞表面受体的缺陷4、转录因子5、受损信号通路6的改变表达、形成的突变激活癌基因7, 或抑癌基因的失活8。
开发了各种方法来研究髓系发育, 并评估了特定基因改变在这一过程中的作用。用于研究 myelopoiesis 的常用方法涉及原代细胞和转基因小鼠。虽然这些模型允许获取生物相关的数据, 但它们有一定的局限性。使用初级细胞遇到有限的细胞数量和限制的文化周期, 缩小改变基因表达和随后的生物或生化分析的可能性。转基因小鼠成本高昂, 需要合理程度的生物合理性。此外, 使用体内模型也增加了一定程度的复杂性, 从而理解了在给定过程中感兴趣的基因的作用。因此, 需要采取其他办法来规避这些限制。细胞系具有无可争辩的优势: (1) 它们具有无限的增殖能力, 允许产生足够的物质进行生物化学和生物研究, (2) 它们容易受到基因的操控 (击倒, 挖空,过度表达), (3) 成本相对较低, (4) 它们允许某些实验方法所需的生物简化程度。
父母 IL-3 (Interleukin-3) 依赖32D 细胞系成立于1983年由 Greenberger 和同事通过感染的骨髓细胞从 C3H/HeJ 鼠与朋友小鼠白血病病毒9。文献中描述了几个32D 克隆: cl-239、cl-310和 cl-1011。32D cl-3 细胞在 IL-3 中增殖, 并经粒细胞集落刺激因子 (CSF)10治疗中性分化。相反, 32D cl-10 细胞, 而 IL-3 依赖, 最初并没有区别于对 G 脑脊液治疗的反应。在 1995年, Touw 博士 retrovirally 转基因 32D cl-10 细胞与野生类型和突变形式的 g-脑脊液受体 (g 脑脊液 R), 以确定该受体的功能重要区域11。这项研究结果产生的32维/克脑脊液 R 细胞, 这是同样依赖于 IL-3, 但在6至10天后, 替换 IL-3 与 G 脑脊液, 细胞停止增殖和不可逆转地分化成成熟的中性粒细胞。这些特性使 32D cl-3 和32维/克脑脊液 R 细胞简化模型的小鼠中性分化, 可由两个明确的增长和分化因子-IL-3 和 G 脑脊液。在过去的几十年中, 多组使用了32维/克脑脊液 R 细胞来研究特定基因在培养过程中细胞增殖和分化中的作用12,13,14,15,16, 并研究 G CSF 信号17,18。重要的是, 使用该细胞线获得的结果与主要细胞和转基因小鼠的数据相关,16,19,20,21。因此, 我们认为, 32 维/克脑脊液 R 细胞作为一种广泛使用和建立良好的模型, 是研究髓样分化的一个有价值的系统, 可与处理这个问题的其他方法并行使用。
在这里, 详细的协议描述处理32维/克-脑脊液 R 细胞线, 包括扩展, 分化, 并评估这些细胞的增殖和分化。提供了32维/克-脑脊液 R 细胞的基因修饰的详细信息, 可以采用逆转录病毒或慢病毒载体转导, 以及病毒滴定的协议。此外, 还提供了一些表明潜在应用32维/克-脑脊液 R 细胞的典型结果。
Protocol
注: 以下是描述32维/克-脑脊液 R 细胞的扩张、分化和转导的步骤。 1. 准备 媒体准备 准备250毫升培养基: RPMI (罗斯威尔公园纪念研究所) 1640 中辅以10% 种热灭活 (胎牛血清) 和小鼠 IL-3 (10 ng/毫升)。 或者, 使用自制的 IL-3。生产国产 IL-3, 传感器 HEK293 细胞 IL-3 表达载体和收集 IL-3 含有上清22。注: 抗生素, 如青霉素 G (100 毫升/ml…
Representative Results
32维/G-脑脊液 R 细胞的增殖与分化 为评估32维/g-脑脊液 r 细胞在促增殖和诱导分化条件下的增殖, 分别在含有 IL-3 和脑脊液的培养基中培养出32维/克脑脊液 r 细胞。据观察, 在含有培养基的 IL-3 中培养的细胞 (10 ng/毫升) 大约每 24 h (图 2A) 分。在更换 IL-3 时 (100 ng/毫升) 的增殖逐渐减慢并在4天后停止 (<strong clas…
Discussion
实验模型的选择是研究的主要课题之一。虽然主要动物和人类细胞被认为能产生最具生物学意义的数据, 但这些模型可能涉及伦理问题, 往往与昂贵和/或复杂的隔离/培养程序相关。初级细胞数量有限, 很难进行基因调控。此外, 主单元格表示由各种单元格类型组成的异构填充, 可能会使数据解释29复杂化。相反, 细胞线提供了一个几乎无限的生物材料来源, 是成本效益, 并允许绕过?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢 Delwel 教授和 Touw 教授为我们提供了32维/克脑脊液 R 细胞线, 丹尼尔教授 Tenen 为我们提供了 Bosc23 细胞系。这项工作得到了捷克共和国赠款机构 (GACR 15-03796S 和 GACR 17-02177S) 赠款的支助, 由捷克科学院分子遗传学研究所 (RVO 68378050) 提供的支助, 以 ma j, 一项 GA 英国研究金 (341015 号项目)。从布拉格的查尔斯大学到 MK, 和一个 GA 英国奖学金 (1278217 号项目) 从布拉格查尔斯大学到 PD。
Materials
| RPMI 1640 powder medium | Merck, Kenilworth, NJ, USA | T 121-10 | without NaHCO3, with L-glutamine |
| DMEM | Thermo Fisher Scientific, Waltham, MA, USA | 15028 | |
| Opti-MEM I Reduced Serum Medium | Thermo Fisher Scientific, Waltham, MA, USA | 31985-047 | L-Glutamine, Phenol Red |
| Fetal bovine serum (FBS) | PAA Laboratories (GE Healthcare,Chicago, IL, USA) | MT35011CV | For differentiation of 32D/G-CSF-R cells |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific, Waltham, MA, USA | 10270 | Used for culturing HEK293T, NIH3T3, BOSC23 cells |
| Penicillin | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | P3032 | |
| Streptomycin | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | S9137 | Streptomycin sulfate salt powder |
| Gentamicin | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | G1914 | |
| murine IL-3 | PeproTech, Rocky Hill, NJ, USA | 213-13 | |
| human G-CSF | PeproTech, Rocky Hill, NJ, USA | 300-23 | |
| Polyethylenimine | Polyscience, Warrington, PA, USA | 23966 | Linear, MW 25,000 (PEI 25000) |
| Polybrene | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | H9268 | |
| Trypsin | VWR Chemicals, Radnor, PA, USA | 0458 | |
| EDTA | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | E5134 | |
| Crystal violet | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | C0775 | |
| Trypan blue | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | T6146 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | D2650 | |
| May-Grünwald Giemsa | DiaPath, Martinengo, BG, Italy | 10802 |
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Citar este artículo
Zjablovskaja, P., Danek, P., Kardosova, M., Alberich-Jorda, M. Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells. J. Vis. Exp. (132), e57033, doi:10.3791/57033 (2018).