体外骨髓源性巨噬细胞对髓鞘碎片的吞噬
Summary
本文采用荧光标记髓鞘碎片和胞内脂滴染色的方法, 对小鼠骨髓源性巨噬细胞的吞噬能力进行评估。
Abstract
骨髓源性巨噬细胞 (BMDMs) 是成熟的白细胞, 具有重要的生理作用, 作为专业吞噬能够清除各种微粒。正常情况下, BMDMs 受中枢神经系统 (CNS) 的限制, 但在受伤后, 他们可以很容易地渗入。一旦在受损的中枢神经组织内, BMDMs 是主要的细胞类型, 负责清除损伤衍生的细胞碎片, 包括大量的富含脂质的髓鞘碎片。中枢神经系统内 BMDM 浸润和髓鞘碎片吞噬的病理影响是复杂的, 不太清楚。这里描述的协议, 允许直接的体外研究 BMDMs 在中枢神经系统损伤的情况下。我们覆盖小鼠 BMDM 分离和培养, 髓鞘碎片制备, 并测定 BMDM 髓鞘碎片吞噬功能。这些技术在不需要重要的专用设备或材料的情况下产生可靠的可量化结果, 但可以很容易地定制以满足研究人员的需要。
Introduction
骨髓源性巨噬细胞 (BMDMs) 是先天和适应性免疫系统之间的重要联系。作为抗原呈递细胞 (apc), 他们可以通过抗原呈递和细胞因子释放1,2,3与淋巴细胞进行通信。然而, 作为专业吞噬, 他们的主要功能是清除病原体, 衰老细胞和细胞碎片1,4。继脊髓损伤 (SCI), 大量髓鞘碎片是由死亡的突, 细胞类型负责中枢神经系统轴突鞘5。我们和其他人已经表明, 清除髓鞘碎片主要是渗透 BMDMs 的责任5,6,7。然而, 在脊髓损伤部位吞没髓鞘碎片被建议将这些正常的抗炎细胞转变为炎状态5,8,9。BMDMs 是脊髓损伤中神经炎症的重要介质, 是临床的主要靶点。
为了帮助研究 BMDMs 在损伤脊髓中的影响, 我们开发了一个体外模型, 直接探讨 BMDMs 对髓鞘碎片的反应。为了提高生物学相关性, 在这些调查中使用了小鼠 BMDMs 和新近分离的髓鞘碎片。因此, 这里介绍的方法也详述了原小鼠 BMDMs 的分离和培养, 以及用于分离鼠中枢神经系统衍生髓鞘碎片的改良蔗糖梯度技术10,11,12。髓鞘碎片可以很容易地标记为荧光染料, 羧基琥珀酰亚胺酯 (CFSE), 以跟踪其内部化的 BMDMs. CFSE 是非常适合这个应用, 因为它是 non-cytotoxic, 其狭窄的荧光光谱允许多路传输与其他荧光探针13,14。继吞噬后, 髓鞘碎片脂通过溶酶体转运, 并以中性脂质包装成细胞内脂滴5。为了量化这种细胞内脂质的积累, 我们提出了一个油红 O (破产) 染色方法优化定量图像分析。这个简单的染色方法产生可靠的重现性结果和量化的15。这些方法有助于研究的髓鞘碎片吞噬和脂质保留与有限的专用设备。
Protocol
Representative Results
Discussion
这里描述的程序利用了新分离的原始的中枢神经系统髓鞘碎片和原发骨髓巨噬细胞。为了减少动物的开支, 我们建议在牺牲时从每只老鼠身上采集大脑和骨髓细胞。两位研究人员一起工作可以同时准备两种材料。另外, 在-80 ° c 的 PBS 中, 大脑可以在髓鞘碎片隔离之前用抗生素补充。我们的经验是, 大脑可以在这些条件下维持1月, 而不会有明显的髓鞘碎片生成潜能的损失。
CFSE ?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者想感谢苏联医学院的媒体专家, 他在视频制作、编辑和配音方面的工作。
这项工作得到了国家卫生研究院 (R01GM100474 和 R01GM072611) 的支持。
Materials
| DMEM | GE Healthcare Life Sciences | SH30243.01 | High glucose with L-glutamine, sodium pyruvate |
| Penicillin-Streptomycin Solution | Corning | 30-002-CI | 100X Solution |
| New Born Calf Serum (NCS) | Rocky Mountain Biologics | NBS-BBT-5XM | United States Origin |
| NCTC clone 929 [L cell, L-929, derivative of Strain L] | ATCC | CCL-1 | L929 Cell Line of Conditioned Media Preparation |
| 24-well Cell Culture Plates | VWR | 10062-896 | |
| Cell Culture Dish | Greiner Bio-One | 639960 | Polystyrine, 145/20mm |
| CFSE Cell Proliferation Kit | Thermo Fisher | C34570 | DMSO for Reconsitution Provided |
| Fluoro-gel with Tris Buffer | Electron Microscopy Sciences | 17985-11 | |
| Oil Red O | Sigma Aldrich | O0625 | |
| Equipment | |||
| Materials | Company | Catalog Number | Comments |
| Ultracentrifuge Tubes | Beckman Coulter | 326823 | Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm |
| SW 32 Ti Ultracentrifuge Rotor | Beckman Coulter | 369650 | SW 32 Ti Rotor, Swinging Bucket, Titanium |
| Hand Held Rotary Homogenizer | Fisher Science | 08-451-71 |
Referenzen
- Wynn, T. A., Chawla, A., Pollard, J. W. Macrophage biology in development, homeostasis and disease. Nature. 496 (7446), 445-455 (2013).
- Unanue, E. R. Antigen-presenting function of the macrophage. Annu Rev Immunol. 2, 395-428 (1984).
- Cavaillon, J. M. Cytokines and macrophages. Biomed Pharmacother. 48 (10), 445-453 (1994).
- Ren, Y., Savill, J. Apoptosis: the importance of being eaten. Cell Death Differ. 5 (7), 563-568 (1998).
- Wang, X., et al. Macrophages in spinal cord injury: phenotypic and functional change from exposure to myelin debris. Glia. 63 (4), 635-651 (2015).
- Goodrum, J. F., Earnhardt, T., Goines, N., Bouldin, T. W. Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study. J Neurosci. 14 (1), 357-367 (1994).
- Greenhalgh, A. D., David, S. Differences in the Phagocytic Response of Microglia and Peripheral Macrophages after Spinal Cord Injury and Its Effects on Cell Death. J Neurosci. 34 (18), 6316 (2014).
- Sun, X., et al. Myelin activates FAK/Akt/NF-kappaB pathways and provokes CR3-dependent inflammatory response in murine system. PLoS One. 5 (2), e9380 (2010).
- Gensel, J. C., Zhang, B. Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res. , 1-11 (2015).
- Bourre, J. M., Pollet, S., Daudu, O., Le Saux, F., Baumann, N. Myelin consists of a continuum of particles of different density with varying lipid composition: major differences are found between normal mice and quaking mutants. Biochimie. 59 (10), 819-824 (1977).
- Larocca, J. N., Norton, W. T. . Current Protocols in Cell Biology. , (2001).
- Norton, W. T., Poduslo, S. E. Myelination in rat brain: method of myelin isolation. J Neurochem. 21 (4), 749-757 (1973).
- Wang, X. Q., Duan, X. M., Liu, L. H., Fang, Y. Q., Tan, Y. Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling. Acta Biochim Biophys Sin (Shanghai). 37 (6), 379-385 (2005).
- Parish, C. R. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol. 77 (6), 499-508 (1999).
- Deutsch, M. J., Schriever, S. C., Roscher, A. A., Ensenauer, R. Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells. Anal Biochem. 445, 87-89 (2014).
- Kroner, A., et al. TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord. Neuron. 83 (5), 1098-1116 (2014).
- Trouplin, V., et al. Marrow-derived Macrophage Production. J Vis Exp. (81), e50966 (2013).
- Chen, S., So, E. C., Strome, S. E., Zhang, X. Impact of Detachment Methods on M2 Macrophage Phenotype and Function. J Immunol Methods. 426, 56-61 (2015).
- Guo, L., et al. Rescuing macrophage normal function in spinal cord injury with embryonic stem cell conditioned media. Mol Brain. 9 (1), 48 (2016).
- Bosco, D. B., et al. A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis. PLOS ONE. 12 (2), e0172925 (2017).
- Ramírez-Zacarías, J. L., Castro-Muñozledo, F., Kuri-Harcuch, W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O. Histochem. 97 (6), 493-497 (1992).
- Koopman, R., Schaart, G., Hesselink, M. K. Optimisation of oil red staining permits combination with immunofluorescence and automated quantification of lipids. Histochem Cell Biol. 116 (1), 63-68 (2001).
- Fang, H., et al. Lipopolysaccharide-Induced Macrophage Inflammatory Response Is Regulated by SHIP. J Immunol. 173 (1), (2004).
- Martinez, F. O., Sica, A., Mantovani, A., Locati, M. Macrophage activation and polarization. Front Biosci. 13, (2008).
- Mosser, D. M., Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 8 (12), 958-969 (2008).
