受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化
Summary
该协议允许研究员分离和表征组织驻留巨噬细胞在各种标志发炎组织提取从饮食诱发的代谢紊乱模型。
Abstract
肥胖促进了一种慢性炎症状态, 主要由组织驻留的巨噬细胞以及单核细胞衍生的巨噬病介导。饮食诱发肥胖 (迪奥) 是研究巨噬细胞异质性作用的有价值的模型;然而, 足够的巨噬细胞隔离很难从发炎的组织中获得。在本协议中, 我们概述了从我们的研究获得一个合适的人群组织驻留巨噬细胞后18周高脂肪 (风) 或高脂肪/胆固醇的隔离步骤和必要的故障排除准则 (HFHCD) 饮食干预。本议定书的重点是三标志性组织研究的肥胖和动脉粥样硬化, 包括肝脏, 白脂肪组织 (笏), 和主动脉。我们强调流式细胞术的二元用法如何实现组织驻留巨噬细胞的分离和表征的新维度。本议定书的一个基本部分解决复杂的基础组织特定的酶消解和巨噬细胞分离, 以及随后的细胞表面抗体染色流细胞分析。这项协议解决了现有的复杂的荧光活化细胞分类 (资产管制), 并提出了这些复杂性的澄清, 以获得广泛的范围, 从适当的排序细胞群体的描述。可选的浓缩方法包括对细胞进行分类, 例如稠密的肝脏, 允许在与外地资产管理体系合作时具有灵活性和时间性。简言之, 这项协议帮助研究者评估了一项研究中大量炎症组织的巨噬细胞异质性, 并提供了有洞察力的诊断技巧, 以利于细胞的分离和鉴定。免疫细胞在迪奥介导的炎症。
Introduction
小鼠模型已被广泛用于研究人类疾病的动态。从患病状态的小鼠中正确分离组织驻留细胞可以提供一个平台, 以了解分子和细胞对疾病发病机制的贡献1。肥胖是一种极其重要的疾病。随着胰岛素抵抗和2型糖尿病、心血管疾病和脂肪性肝病的发生, 肥胖的发病率在全球范围内持续上升2,3。通过减少脂肪组织产生的改变信号的体力活动而进一步扭曲了过量的养分消耗, 从而改变了其他外围组织的细胞环境, 如主动脉和肝脏4。这种扰乱代谢稳态导致慢性低等级系统性炎症5。
经典活化的巨噬细胞的主动脉和肝脏, 以及他们的招募白脂肪组织 (笏) 已被证明不仅启动失调的代谢信号, 但也持续炎症6,7。巨噬细胞表型和功能性异质性与肥胖相关疾病的发病机制有密切关系7。巨噬细胞极化的动态可塑性允许这些单元表现出一系列激活的表型, 以协调炎症的进展和分辨率8。虽然经典激活 (M1) 巨噬细胞是牵连炎症的传播, 或者激活 (M2) 巨噬细胞已与分辨率和组织修复相关的9,10。
当机体经历新陈代谢的压力时, 白色脂肪组织异常堆积。膨胀的脂肪组织吸引并保留了深刻改变正常脂肪细胞功能的炎性神经细胞, 以促进胰岛素抵抗、高血糖和最终2型糖尿病、胰岛素抵抗或高血糖11, 12。在平行, 白色脂肪组织改造反应的炎症信号的渗透经典活化 (M1) 脂肪组织巨噬细胞 (atm)13,14。这多器官施加一连串的信号, 出轨正常功能的其他机构, 如主动脉和肝脏4。
肝脏是一个新陈代谢的发电站, 适应的刺激来自附近的失调笏15。肝巨噬细胞或枯否体细胞, 在反应代谢变化, 分泌炎症细胞因子, 改变实质和实质细胞表型和促进组织重塑。肝脂堆积、炎症、细胞外基质沉积、坏死及最终功能丧失伴随着非酒精性脂肪肝引起的广泛肝损害的炎症反应16,17,18。
当身体承受慢性代谢压力19时, 大动脉在动脉壁内积聚脂质, 同时与受损的笏和肝功能相同。动脉脂质积累引发了活化的内皮细胞分泌的趋化因子, 并随后招募单核20。一旦被吸收, 单核细胞就会增殖, 分化, 摄取脂蛋白, 成为泡沫。动脉粥样硬化的启动和持续的炎活动的招募和组织居民脂质巨噬细胞。这些巨噬细胞屈服于动脉粥样硬化微环境中的胞外和胞内压力信号, 然后参与凋亡信号级联。由于这些泡沫细胞死亡, 他们贡献了他们的脂质填充内容的坏死核心病变, 然后导致斑块破裂, 心肌梗塞, 中风。
整体而言, 巨噬细胞表型的异质性协调在失调组织中观察到的炎症性改变引起的肥胖, 如笏、肝和主动脉8,21。特征的招募和组织驻留巨噬细胞可以提供洞察的潜在分子目标, 操纵巨噬细胞表型1。为了有效地描述由肥胖引起的炎症组织的巨噬细胞, 可以通过酶消化得到单细胞悬浮液。这种离解协议必须有效地在充分降解结缔组织, 同时尽量减少免疫细胞死亡和提供最佳的细胞产量。酶的混合物取决于组织的类型和它的结构构成。弹性组织, 如主动脉, 需要更强的酶活性, 比肝脏和笏, 以实现组织离解。从单细胞悬浮, 组织驻留巨噬细胞可以明确的特征或孤立的进一步的下游分析, 如转录剖面。
在这里, 一个组织特异性的协议描述, 使用胶原酶相关的组织消化和多色流式细胞仪有效分离和表征组织驻留巨噬细胞从传统的饮食诱发肥胖,动脉粥样硬化, 简单的脂肪和脂肪小鼠模型。同时染色细胞表面标记抗体抗白细胞-(CD45 和/或 CD11b) 和巨噬细胞 (F4/80) 特异抗原通常用于识别巨噬细胞的数量22。荧光活化细胞分类 () 是一种强有力的策略, 用来对这些被识别的种群进行高纯度的分类。然后利用下游分子分析 (如定量实时聚合酶链反应)23对表型特定基因图谱进行排序。虽然标准流式细胞仪和流 cytometry-based 细胞排序是强大的工具, 区分巨噬细胞在一个巨大的异构电池悬浮, 前协议必须优化, 以确保成功的输出。在本研究中, 描述了有效分离和表征可行组织特异性巨噬细胞的协议;更重要的是, 这项研究提供了对经常出现的技术问题的重要洞察力, 以及预防和/或解决它们的主动和 trouble-shooting 战略。
Protocol
Representative Results
Discussion
饮食诱导代谢紊乱模型, 模仿疾病, 如动脉粥样硬化, 简单的脂肪, 脂肪性肝炎和2型糖尿病广泛使用, 以更好地了解潜在的分子机制的疾病进展。胶原酶依赖性消化经常被用于游离组织, 从细胞外基质 (ECM)16,27中释放出癌细胞。酶, 如胶原蛋白酶扰乱胶原蛋白, 为相邻细胞提供结构支持。组织的结构组成决定了组织基质的刚度 (抵抗变形), 哪些粗胶原酶产品?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢宾夕法尼亚州立大学千禧科学综合体的流式细胞仪核心设施。
Materials
| 26G x 5/8 in Needles | BD | 305115 | |
| 23G x 0.75 in needle x 12 in. tubing Blood Collection Set | BD | 367297 | Used for cannulation of subhepatic IVC during liver perfusion |
| 21G 1 1/2 in. Needles | BD | 305156 | |
| 1mL syringe with rubber stops | BD | 309659 | |
| 10mL Syringes | BD | 309604 | |
| 1mL Syringe | BD | 309659 | |
| F4/80 PE | Biolegend | 123110 | |
| CD11c PE/Cy7 | Biolegend | 117318 | |
| CD11b PE/Cy5 | eBioscience | 15-0112-81 | |
| Anti-mouse CD16/32 Fc Block | Biolegend | 101320 | |
| CD45 Pacific Blue | Biolegend | 103126 | |
| PE Rat IgG2a | Biolegend | 400508 | |
| PE/Cy7 Armenian Hamster IgG | Biolegend | 400922 | |
| PE/Cy5 Rat IgG2b | Biolegend | 400610 | |
| Pacific Blue Rat IgG2c | Biolegend | 400717 | |
| Dulbecco’s Modified Eagle Medium (DMEM) | Cellgro | 15-017-CV | |
| 1X Phosphate Buffered Saline (PBS) | Cellgro | 21-031-CV | |
| 70 micron cell strainers | Corning, Inc. | 352350 | |
| 1.7 mL microcentrifuge tube | Denville | C2170 | |
| Paraformaldehyde Aqueous Solution -16X | Electron Microscopy Sciences | CAS #30525-89-4 | |
| Micro Dissecting Scissors, 3.5 inch, Straight, 23mm, Sharp | Stoelting | 52132-10P | Used for general dissecting purposes |
| Micro Forceps, 4in, full curve, 0.8mm | Stoelting | 52102-37P | Used for general dissecting purposes |
| Spring dissection scissors – 3 mm Cutting edge | Fine Science Tool | 15000-10 | Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28 |
| Curved 0.07 x 0.04 mm Tip Forceps | Fine Science Tool | 11297-10 | Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28 |
| Hemostatic Forceps (Curved) | Fine Science Tool | 13021-12 | |
| Heparin Sodium Salt | Fischer Scientific | 9041-08-1 | |
| 35mm Cell Culture/Petri Dishes | Fischer Scientific | 12-565-90 | |
| Polystyrene Petri Dishes (10 cm) w/lid | Fischer Scientific | 08-757-100D | |
| 15mL Conical Centrifuge Tubes (Polypropylene) | Fischer Scientific | 14-959-53A | |
| 50mL Conical Centrifuge Tubes (Polypropylene) | Fischer Scientific | 14-432-22 | |
| 5mL Round-Bottom Polystyrene Tubes | Fischer Scientific | 14-959-5 | |
| Fetal Bovine Serum | Gemini Bio-Products | 100-106 | |
| Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous) | Millipore | EX0285-1 | |
| Bovine Serum Albumin | Rockland | BSA-50 | |
| HEPES | Sigma-Aldrich | H3375 | |
| Collagenase Type II | Sigma-Aldrich | C6885 | |
| Collagenasse Type XI | Sigma-Aldrich | C7657 | |
| Hyaluronidase Type I | Sigma-Aldrich | H3506 | |
| DNAse | Sigma-Aldrich | DN25 | |
| Collagenase Type I | Sigma-Aldrich | C0130 | |
| NaOH | Sigma Aldrich | 1310-73-2 | |
| CaCl2 | Sigma Aldrich | 449709-10G | |
| 500mL beaker | Sigma Aldrich | 02-540M | |
| 4 cm Hemostatic clamp | Stoelting | 52120-40 | |
| Toothed forceps | Stoelting | 52104-33P | |
| 50 micron Disposable filters | Systemex | 04-0042-2317 | |
| Collagenase Type IV | ThermoFischer Scientific | 17104019 | |
| Ammonium Chloride Potassium (ACK) | ThermoFischer Scientific | A1049201 | |
| Razors (0.22 mm (0.009")) | VWR International | 55411-050 | |
| Texas Red Live/Dead stain | Red viability stain (in Figure 1A) |
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