检测小鼠中性粒细胞的迁移和渗透
Summary
在这里，我们提出了三种方法，以评估嗜中性粒细胞迁移和渗透在体内和体外。这些方法可用于发现针对中性粒细胞迁移的有前途的治疗方法。
Abstract
嗜中性粒细胞是先天免疫系统的主要成员，在宿主防御病原体和病理炎症反应方面发挥着关键作用。中性粒细胞可以通过细胞因子和化学因子的指导被招募到炎症部位。中性粒细胞的渗透可能导致不分青红皂白的组织损伤，如风湿性关节炎（RA）。从围肠渗出物分离的嗜中性粒细胞对定义的化学吸引剂N-form-Met-Leu-Phe（fMLP）有反应，在Transwell或Zigmond室测定中体外。气囊实验可用于评估中性粒细胞对体内脂多糖（LPS）的化学性评价。佐剂诱发关节炎（AA）小鼠模型常用于RA研究，与抗骨髓过氧化物酶（MPO）或抗中性粒细胞酶（NE）抗体的关节部分的免疫组织化学染色是一种公认的测量方法中性粒细胞渗透。这些方法可用于发现针对中性粒细胞迁移的有前途的疗法。
Introduction
嗜中性粒细胞是最丰富的白细胞，占人类白细胞总体的50-70%。中性粒细胞是急性炎症的主要反应者之一。中性粒细胞可以通过组织驻留细胞2、3、4释放的细胞因子和化学因子的引导，被招募到炎症部位，由中性粒细胞表面的细胞粘附分子和血管内皮细胞5的相互作用来调节。中性粒细胞是宿主防御的基础，并在病理炎症反应中发挥作用，因为它们强大的能力通过释放活性氧物种（ROS）和其他破坏组织分子3，6来破坏组织。
先前的研究描述了小鼠或人类的几种中性粒细胞分离方案。Oh等人演示了一种密度梯度分离方法，将人类嗜中性粒细胞从全人类血液中分离出来7。然而，由于血量小，很难从小鼠血液中分离足够的嗜中性粒细胞。另外，可以从小鼠嗜虫液中引出大量纯和可行的小鼠中性粒细胞，这些纯化的嗜中性粒细胞可用于检查细胞外功能的几个方面，包括嗜中性粒细胞渗透、迁移、化学毒杆菌、氧化爆裂、细胞因子和中性粒细胞外陷阱（NET）生产8。Transwell测定9或Zigmond室测定10，11可用于评估嗜中性粒细胞在体外迁移。气囊模型用于评估嗜中性粒细胞在体内的迁移和渗透。皮下气囊模型是研究炎症细胞迁移的方便体内动物模型。
传统上，中性粒细胞被认为是炎症急性阶段的病原体消除剂。然而，最近的发现表明，嗜中性粒细胞是复杂的细胞，执行大量的特殊功能。嗜中性粒细胞可以调节许多过程，如急性损伤和修复，肿瘤发生，自身免疫反应，和慢性炎症12，13。中性粒细胞还调节适应性免疫反应，并能调节B细胞和T细胞14，15。中性粒细胞的大量短缺导致人类死亡或严重的免疫缺陷，小鼠的嗜中性粒细胞耗竭导致死亡，而器官中中性粒细胞的过度激活或招募会导致多种免疫疾病，如风湿性关节炎（RA）和系统性红斑狼疮（SLE）6。嗜中性粒细胞是RA患者的阴膜流体中最丰富的细胞。嗜中性粒细胞通过降解产生过量的骨髓过氧化物酶（MPO）和中性粒细胞酶（NE），从而加剧软骨侵蚀。MPO是一种过氧化物酶，主要表现在嗜中性粒细胞16的颗粒中。NE与关节软骨破坏有关17。MPO和NE可用于评估嗜中性粒细胞迁移和在RA患者组织中渗透的状态。
本文提供了三种常规方法，用于评估在体内和体外诱导的正常嗜中性粒细胞的迁移，以及病理性嗜中性粒细胞在小鼠关节特异性炎症模型中的渗透。
Protocol
所有实验程序均经北京中医药大学动物护理与使用委员会审核通过。 注：使用C57BL/6小鼠（7-8周大）。 1. 中性粒细胞分离 围肠渗出细胞的采集 在 ddH2O 中制备新鲜的 10% 蛋白酶肽溶液。根据小鼠数量计算所需的体积。注：将小鼠数量设置为N，（2N+1）溶液的mL必须提前溶解和过滤。 用 70% 乙醇喷洒工作空间。使用胰岛素注射…
Representative Results
从小鼠的洗漱液中收集围渗出细胞。细胞在 1 mL 的 RPMI-1640 完整介质中重新悬浮，分层到两步 （54.8%/70.2%）不连续密度梯度（图1A），在1，500 x g下离心30分钟。 嗜中性粒细胞（+95%，±1 x 107嗜中性粒细胞/小鼠）从下界面中恢复（图1B）。 进行?…
Discussion
从外周血7，骨髓和组织18高度纯化的中微粒细胞的详细方案已经提供很长时间。在这里，我们采用一种方法，将嗜中性粒细胞与围肠液19分离，其中成熟的嗜中性粒细胞保持灭活，以便进一步抗炎和抗氧化研究。
我们使用气囊实验来探索LPS诱导的嗜中性粒细胞在体内的渗透。该方法被提出作为直接测量细胞在体内一般炎症…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（助学金号81430099和31500704）、国际合作与交流项目（赠款号2014DFA32950）和北京中医药大学（捐赠）的支持。批号 BUCM-2019-JCRC006 和 2019-JYB-TD013）。
Materials
| 0.1% Fast Green Solution | Solarbio | 8348b | Transfer 20 mg of fast grene FCF in one vial into another 100 mL beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% fast green solution, and filter it using a Nalgene PES 75mm filter |
| 0.1% Safranin O Staining Solution | Solarbio | 8348a | Transfer 20 mg of safranin O stain in one vial into a 100 ml beaker. Add 20 mL of H2O into the beaker and dissolve the stain by stirring to make 0.1% safranin O staining solution, and filter it using a Nalgene PES 75mm filter |
| 0.5 M Ethylenediaminetetraacetic acid solution (EDTA), pH 8.0 | Sigma | 324506 | Sterile |
| 100% Ethanol | Beijing Chemical Works | ||
| 100% Methanol | Beijing Chemical Works | ||
| 15 mL Conical Polypropylene Centrifuge Tube | Falcon | 14-959-53A | |
| 23 G x 1 1/4" Needle | BD | 305120 | |
| 26 G x 3/8" Needle | BD | 305110 | |
| 3% bovine serum albumin (BSA) | Dissolve 0.3 g BSA in 10 mL PBS | ||
| 3% H2O2 | Mix 1 mL 30% H2O2 with methanol with 9 mL methanol | ||
| 3,3'-diaminobenzidine (DAB) kit | ZSGB-BIO | ZLI-9018 | |
| 30 G x 1/2" Needle | BD | 305106 | |
| 30% H2O2 | Beijing Chemical Works | ||
| 5 mL Syringe | BD | Z683574 | |
| 50 mL Conical Polypropylene Centrifuge Tube | Falcon | 14-432-22 | |
| 50% Ethanol | Mix 500 mL 100% ethanol with 500 mL dH2O | ||
| 54.8% Percoll | Mix 2.74 mL SIP with 2.26 mL 1×PBS, stand still | ||
| 70% Ethanol | Mix 700 mL 100% ethanol with 300 mL dH2O | ||
| 70.2% Percoll | Mix 3.51 mL SIP with 1.49 mL 1×PBS, stand still | ||
| 80% Ethanol | Mix 800 mL 100% ethanol with 200 mL dH2O | ||
| 95% Ethanol | Mix 950 mL 100% ethanol with 50 mL dH2O | ||
| Acid Alcohol Superfast Differentiation Solution | Beyotime | C0165S | |
| ANTIBODIES | |||
| Anti-Myeloperoxidase Antibody | Abcam | ab208670 | |
| Anti-Neutrophil Elastase Antibody | Abcam | ab21595 | |
| Automatic Hematology Analyzer | Sysmex | XS-800i | |
| Bovine Serum Albumin (BSA) | VWR | 0332-100G | |
| Complete Freund's Adjuvant, 10 mg/ml | sigma | 1002036152 | |
| Cover Slip | CITOGLAS | 10212432C | |
| Dial Thickness Gauge | Mitutoyo | 7301 | |
| Eppendorf Microtubes, 1.5 mL | Sigma | Z606340 | |
| Foetal Bovine Serum (FBS) Premium | PAN | P30-1302 | |
| Gas Anesthesia System | ZS Dichuang | ZS-MV-IV | |
| Goat Anti-Rabbit IgG H&L (HRP) | PPLYGEN | C1309 | This is the secondary antibody used in the immunohistochemical staining. |
| Hank's Balanced Salt Solution (HBSS) | Biological Industries, Beth HaEmek, Israel | 02-016-1A | Sterile |
| Hematoxylin Staining Solution | ZSGB-BIO | ZLI-9609 | |
| Lipopolysaccharide (LPS) | Sigma | L3012 | |
| MEDIA AND SUPPLEMENTS | |||
| Modified Safranin O-fast Green FCF Cartilage Stain Kit | Solarbio | G1371 | |
| N-formyl-Met-Leu-Phe (fMLP) | Sigma | 47729 | |
| Penicillin Streptomycin Solution, 100× | Invitrogen | 1514022 | |
| Percoll | GE Healthcare | 10245207 | Density gradient medium |
| Permeabilization Buffer | Mix 100 μL Triton X-100 with 1 L dH2O to get 0.01% Triton X-100 | ||
| Phosphate Buffer Saline (PBS), 1× | Mix 90% ddH2O with 10% (v/v) 10×PBS, autoclaved | ||
| Phosphate Buffer Saline (PBS), 10× | Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na2HPO4·2H2O, 0.48 g KH2PO4 (anhydrous) in 200 mL ddH2O, adjust pH 7.4, autoclaved | ||
| PLASTIC WARES AND EQUIPMENTS | |||
| POWDER | |||
| Proteose Peptone | Oxoid | 1865317 | |
| Retrieval Buffer | Mix 18 mL retrieval buffer A with 82 mL retrieval buffer B, add dH2O to 1000 mL, adjust pH to 6.0 | ||
| Retrieval Buffer A Stock for IHC | Dissolve 4.2 g citric acid (C6H5O7·H2O) in 200 mL dH2O | ||
| Retrieval Buffer B Stock for IHC | Dissolve 5.88 g trisodium citrate dihydrate (C6H5Na3O7·2H20) in 200 mL dH2O | ||
| Roswell Park Memorial Institute (RPMI)-1640 medium | Sigma | R8758 | |
| RPMI-1640 Complete Medium | RPMI-1640 medium is supplemented with 10% FBS and 1% penicillin/streptomycin. | ||
| Shu Rui U40 Disposable Sterile Insulin Injection Needle 1 mL | BD | 328421 | |
| Slide | CITOGLAS | 10127105P-G | |
| SOLUTION | |||
| Stock Isotonic Percoll (SIP) | Mix 90% (v/v) of percoll with 10% (v/v) 10×PBS, stand still for 20 min | ||
| Wash Buffer in Air Pouch Assay | Dilute 0.5M EDTA to 10mM with HBSS | ||
| Xylene | Beijing Chemical Works |
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Citar este artigo
Lu, Q., Yuan, K., Li, X., Jiang, H., Huo, G., Jia, W., Huang, G., Xu, A. Detecting Migration and Infiltration of Neutrophils in Mice. J. Vis. Exp. (156), e60543, doi:10.3791/60543 (2020).