巨噬细胞通过二维电泳蛋白组学分析
Summary
Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.
Abstract
The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.
Introduction
巨噬细胞是异质的，塑料的细胞，能够获得不同的功能表型。 在体内，这些细胞对大量的各种微环境signalssuch作为微生物产物，细胞因子， 等等 。1的响应。 在体外 ，所述促炎性表型（M1）的巨噬细胞可通过脂多糖（LPS）和抗炎表型（M2），通过某些细胞因子，如白介素-4（IL-4）诱导。此外，巨噬细胞可以从一个激活的M1至M2的表型转换，而相反地，在特定的信号2。
根据不同的表型，巨噬细胞将具有不同的功能。 M1巨噬细胞产生促炎细胞因子，如肿瘤坏死因子α（TNF-α），杀灭微生物或肿瘤细胞的3个细胞。与此相反，M2巨噬细胞阻止这些炎性应答状在伤口愈合和纤维化由产生抗inflammatorÝ因子如TGF-β3,4。
从健康供体的人外周血单核细胞通过Ficoll密度梯度离心法如使用适于从Boyum 6的技术先前描述5中分离得到。在培养的巨噬细胞后6天的原代培养7可以分化成M1或M2的表型。
蛋白质表达或根据各种受控刺激，如宿主的致病性或微生物毒素的巨噬细胞的两种亚型之间的蛋白质变化的分析，将是有益的破译亲和抗炎的巨噬细胞的功能。
蛋白质组学是用于直接监控的蛋白质，是具体地上调或下调的下各种刺激培养的人体巨噬细胞的独特工具。荧光染料已解决了某些二维凝胶电泳的局限性，如低灵敏度和图像分析8 < / SUP>。染料与半胱氨酸残基反应而增加的检测灵敏度相比，那些与赖氨酸残基9的反应。在先前的研究中，我们证明了DIGE饱和标记的有用稀少样品10的分析结果相比较，以经典的银染的二维电泳11。这种技术是在快速分析巨噬细胞的两种亚型之间或从同一亚型未处理和处理的巨噬细胞之间的蛋白质修饰很有帮助。
此蛋白质组技术的优点是具有由2D凝胶12分析获得的蛋白质的大小和翻译后修饰的信息。应当考虑到，这不是一个高通量技术，限制了可被分析的样品的数目。基于质谱的高通量测定法评论了发展最近13可以改善这一点。
_content“>这里，我们提出了如何通过电泳，等电聚焦的过程中执行从培养的巨噬细胞的蛋白提取2D DIGE分析和SDS-PAGE，以及对适当的2D软件的有用信息。Protocol
Representative Results
Discussion
本文所描述的协议详细说明了分析巨噬细胞中，M1（促炎）和M2（抗炎）的两个亚型的各种刺激的影响的方法。的M1和M2巨噬细胞的原代培养物是从单核细胞分化得到的先前公布的7。
的2D DIGE凝胶电泳的方法需要特殊的材料和设备，如等电聚焦，以扫描在对Cy3和Cy5，一个扫描器，用于荧光激发/发射波长下的凝胶两次细胞对于等电聚焦，低荧光板用于SDS-PAGE上和2D软件。这…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).
Materials
| Name | Company | Catalog number |
| RPMI 1640 | Invitrogen | 31870-074 |
| PBX 10 X | Invitrogen | 14200-083 |
| L-glutamine-200mM-100X | Invitrogen | 25030-024 |
| gentamycin 10 mg/mL | Invitrogen | 15710-049 |
| human serum | Invitrogen | 34005100 |
| Ficoll d=1,077 | ATGC | L6115 |
| Leucosep | Dutscher | 16760 |
| 6-wells plate PRIMARIA | Becton Dickinson | 353846 |
| IL-4 | Promocell | B-61410 |
| lipopolysaccharide | Sigma-Aldrich | L-2654 |
| Giemsa | Fluka | 48900 |
| EDTA MM372,2 | Research Organics | 3.00E+01 |
| Filter 0,22µm | Millipore | SCGPTORE |
| 100 mL cylinder | Corning | 430182 |
| TCEP | Interchim | UP242214 |
| Bradford reagent | Bio-Rad | 5000006 |
| Cy3+Cy5-reactive dye | GE Healthcare | 25-8009-83 |
| IPG strip 3-10 24cm | GE Healthcare | 17-6002-44 |
| Protean IEF cell | Bio-Rad | 165-4000 |
| Low-melting agarose | Invitrogen | 15517-014 |
| Ettan-Daltsix system | GE Healthcare | 80-6485-08 |
| Ettan DIGE Imager scanner | GE Healthcare | |
| Progenesis Samespot | Non linear dynamics | |
| 50 ml tubes | any supplier | n/a |
| 15 ml tubes | any supplier | n/a |
| CHAPS | Sigma-Aldrich | C5070 |
| Urée | Merk | 108484-500 |
| Thiourée | Sigma-Aldrich | T7875 |
| DTT | Bio-Rad | 1610611 |
| APS | Sigma-Aldrich | A3678 |
| TEMED | Sigma-Aldrich | T9281 |
| Tris Base | Sigma-Aldrich | T1503 |
| Tris HCl | Sigma-Aldrich | T3253 |
| Pharmalytes 3-10 | GE Healthcare | 17-0456-01 |
| SDS | Sigma-Aldrich | L3773 |
| Bromophenol blue | Sigma-Aldrich | 114391 |
| Glycerol | Sigma-Aldrich | G6279 |
| Acrylamide 40% | Bio-Rad | 161-0148 |
| 2D clean Up | GE Healthcare | 80-6454-51 |
| Glycine | Sigma-Aldrich | G7126 |
| Diméthylformamide | Sigma-Aldrich | 22705-6 |
| electrode wicks | Bio-Rad | 165-4071 |
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