巨噬细胞报告器细胞分析，以检查聚合物表面的吸附蛋白层上的收费类似受体介导NF-kB/AP-1信号
Summary
该协议为研究人员提供了一种快速、间接的方法，用于测量小鼠巨噬细胞系中与TLR相关的NF-B/AP-1转录因子活性，以响应各种聚合物表面和吸附蛋白层，这些蛋白质层为生物材料植入微环境建模。
Abstract
对植入的生物材料（称为异物反应）的持续炎症宿主反应是生物医学设备和组织工程结构开发和实施方面的一个重大挑战。巨噬细胞，一种先天免疫细胞，是异物反应的关键参与者，因为它们在设备的寿命期间停留在植入部位，并且经常被研究，以理解这种有害的宿主反应。许多生物材料研究人员已经表明，在植入材料上吸附蛋白质层会影响巨噬菌体行为，进而影响宿主反应。本文的方法描述了一种体外模型，利用在聚合物生物材料表面含有细胞损伤分子的吸附蛋白层来评估巨噬细胞反应。NF-*B/AP-1报告器巨噬细胞系和相关色度碱性磷酸酶测定作为一种快速方法，间接检查NF-*B/AP-1转录因子活性，以响应含有血液蛋白和损伤相关分子模式的复杂吸附蛋白层，作为在体内生物材料表面形成的复杂吸附蛋白层的模型。
Introduction
异物反应（FBR）是一种慢性宿主反应，通过持续释放炎症介质和阻碍植入物质与周围组织1的整合，会对植入的物质或装置（如药物输送装置、生物传感器）的性能产生负面影响。这种先天免疫反应是由植入程序引起的，其特点是先天免疫细胞和纤维胶囊在植入物1周围长期存在。在物质宿主响应的上下文中，巨噬菌体-材料相互作用对宿主响应的进展和FBR1的发展有显著影响。巨噬细胞是一种多样化的先天免疫细胞群，从组织居民巨噬细胞群或从血液中提取的单细胞衍生巨噬细胞被招募到植入部位。它们在植入后不久开始在植入部位积累，并在几天内成为植入微环境中的主要细胞群。物质粘附性巨噬细胞，以及通过巨噬细胞融合形成的异体巨细胞（FBGC），可以在植入物2、3的寿命内在材料表面保持。因此，巨噬细胞被认为是异体反应的关键参与者，因为它们的作用是协调FBR的特征步骤：急性炎症反应、组织重塑和纤维化组织形成1。
收费受体（TTL）是一个模式识别受体家族，由许多免疫细胞（包括巨噬细胞）表达，并已被证明在炎症和伤口愈合中发挥重要作用。除了病原体衍生的配体外，TDR 能够结合内源性分子，称为损伤相关分子模式 （DAMPs），这些分子在细胞坏死期间释放并激活炎症信号通路，导致产生亲炎细胞因子4。我们和其他人提出，软组织生物材料植入过程中发生的损伤释放DAMPs，然后吸附到生物材料表面，除了血液蛋白和调节随后的细胞-物质相互作用5，6。当巨噬细胞与植入物上的吸附蛋白层相互作用时，其表面TVR可以识别吸附的DamPs并激活亲炎信号级联，导致NF-βB和AP-1转录因子的激活和原炎细胞因子的产生。我们之前已经表明，与仅吸附血清或血浆表面（即不存在DAMPs）相比，小鼠巨噬细胞显著地增加了NF-β-B/AP-1活性和肿瘤坏死因子α（TNF-α，前激细胞因子）分泌物，以响应各种聚合物表面的含DAMP吸附蛋白层，而仅吸附血清或血浆的表面（即，不存在DAMPs），而这种反应主要是由TLR2调节的。
该协议中使用的NF-+B/AP-1报告宏噬细胞细胞系（材料表）是测量巨噬细胞5、7、8中相对NF-+B和AP-1活性的便捷方法。结合TLR通路抑制剂，该细胞系是研究TLR激活及其在炎症中作用的有用工具，对各种刺激5，7，8。报告细胞是经过修饰的小鼠巨噬细胞样细胞系，可在NF-βB和AP-1转录因子活化9时稳态产生分泌的胚胎碱性磷酸酶（SEAP）。然后，可以使用色度酶碱性磷酸酶测定（材料表）来量化相对量的SEAP表达，作为NF-βB/AP-1活性的间接测量。由于NF-βB和AP-1是许多细胞信号通路的下游，因此可用于验证特定通路的作用，用于验证特定途径的作用，以靶向特定TVR（例如TLR2）或TLR适配器分子（本文中描述的方法提供了一种简单而快速的方法，用于评估 TLR 信号在小鼠巨噬细胞反应中对各种聚合物表面的贡献，其中吸附的蛋白质层同时含有血液蛋白和 DAMPs，作为植入生物材料的体外模型。
Protocol
1. 介质和试剂制备 准备成纤维细胞介质。结合450 mL的Dulbecco的改性鹰培养基（DMEM），50 mL的胎儿牛血清（FBS），和5 mL的青霉素/链霉素。储存在4°C下长达3个月。 准备记者巨噬菌体生长介质在50 mL等分。结合45 mL的DMEM，5 mL的FBS，5μg/mL支原虫消除试剂（材料表）和200μg/mL phleomycin D1（材料表）。储存在4°C下长达3个月。 准备记者巨噬菌体测定介质在50 m…
Representative Results
对聚合物涂层表面的清洁方法进行了测试，以确保涂层不会中断，这被视为水接触角度与未涂布玻璃盖玻片的变化（图2）。在70%乙醇中浸泡PMMA涂层显微镜滑片1小时，可去除PMMA涂层（图2，左侧面板），这可能是由于PMMA在80wt%乙醇13中的溶解性，因此仅使用30分钟的紫外线灭菌就清洗PMMA涂层表面。PMMA涂层的浓度以前已经优化<sup class="xr…
Discussion
我们实验室的一个主要重点是对固体生物材料软组织植入物的宿主响应，特别是植入过程中发生的细胞损伤如何影响宿主响应。这里介绍的初步实验使用报告宏噬细胞细胞系和体外生成的DAMP包含细胞解酶，以研究细胞损伤（即从植入手术）释放的分子对生物材料的巨噬细胞反应的影响。纤维细胞解酶用于模拟由于生物材料放置而导致的细胞损伤和DamPs的释放。由于软组织中成纤维细胞的流行，以?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢加拿大卫生研究院研究项目（PTJ 162251）、皇后大学参议院咨询委员会以及加拿大创新基金会约翰·埃文领导基金（项目34137）和安大略省研究与创新部研究基金（项目34137）提供的基础设施支持。L.A.M.得到了皇后大学R.塞缪尔·麦克劳克林奖学金、加拿大加拿大研究生奖学金硕士学位和安大略省研究生奖学金的支持。作者要感谢Myron Szewczuk博士慷慨地赠送了NF-+B/AP-1报告人大噬细胞系和迈克尔·布伦纳哈塞特博士和桑德拉·洛伦森博士，感谢他们的凝胶成像系统和板阅读器的使用。
Materials
| Cell culture reagents | |||
| anti-mouse/human CD282 (TLR2) | Biolegend | 121802 | |
| CLI-095 (TLR4 inhibitor) | Invivogen | TLRL-CLI95 | |
| C57 complement plasma K2 EDTA 10ml, innovative grade US origin | InnovativeResearch | IGMSC57-K2 EDTA-Compl-10ml | Mouse plasma |
| Dulbecco's modified eagle medium (DMEM) | Sigma Aldrich | D6429-500ML | |
| Dulbecco's phosphate buffered saline (DPBS) | Fisher Scientific | 14190250 | No calcium, no magnesium |
| Fetal bovine serum (FBS), research grade | Wisent | 98150 | |
| LPS-EK | Invivogen | TLRL-EKLPS | Lipopolysaccharide from Escherichia coli K12 |
| NIH/3T3 fibroblasts | ATCC | CRL-1658 | |
| Pam3CSK4 | Invivogen | tlrl-pms | Synthetic triacylated lipopeptide – TLR1/2 ligand |
| Penicillin/streptomycin | Sigma Aldrich | P4333-100ML | |
| Plasmocin | Invivogen | ANT-MPP | Mycoplasma elimination reagent |
| RAW-Blue cells | Invivogen | raw-sp | NF-κB/AP-1 reporter macrophage cell line |
| Trypan blue solution, 0.4% | Fisher Scientific | 15250061 | |
| TrypLE express enzyme (1X) | Fisher Scientific | 12604021 | animal origin-free recombinant cell dissociation enzyme |
| Zeocin | Invivogen | ANT-ZN-1 | |
| Kits and assays | |||
| ELISA precoated plates, mouse IL-6 | Biolegend | B213022 | |
| ELISA precoated plates, mouse TNF-α | Biolegend | B220233 | |
| Endotoxin (Escherichia coli) – Control standard endotoxin (CSE) | Associates of Cape Cope Inc. | E0005-5 | Endotoxin for standard curve in chromogenic endotoxin assay |
| LAL water, 100 mL | Associates of Cape Cope Inc. | WP1001 | Used with chromogenic endotoxin assay |
| Micro BCA protein assay | Fisher Scientific | PI23235 | |
| Limulus amebocyte lysate (LAL) Pyrochrome endotoxin test kit | Associates of Cape Cope Inc. | C1500-5 | Chromogenic endotoxin assay reagent |
| QUANTI-Blue alkaline phosphatase detection medium | Invivogen | rep-qb2 | Alkaline phosphatase assay to indirectly measure NF-κB/AP-1 activity |
| Polymeric coating reagents | |||
| Chloroform, anhydrous | Sigma Aldrich | 288306-1L | |
| Ethyl alcohol anhydrous | Commercial Alcohols | P006EAAN | Sigma: Reagent alcohol, anhydrous, 676829-1L |
| Straight tapered fine tip forceps | Fisher Scientific | 16-100-113 | |
| Fluorinert FC-40 solvent | Sigma Aldrich | F9755-100ML | Fluorinated solvent for fPTFE |
| Cell culture grade water (endotoxin-free) | Fisher Scientific | SH30529LS | |
| Poly(methyl methacrylate) (PMMA) | Sigma Aldrich | 182230-25G | |
| Sylgard 184 elastomer kit | Fisher Scientific | 50822180 | |
| Teflon-AF (fPTFE) | Sigma Aldrich | 469610-1G | Poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] |
| Consumables | |||
| Adhesive plate seals | Fisher Scientific | AB-0580 | |
| Axygen microtubes, 1.5 mL | Fisher Scientific | 14-222-155 | |
| Borosilicate glass scintillation vials, with white polypropylene caps | Fisher Scientific | 03-337-4 | |
| Clear PS 48-well plate | Fisher Scientific | 08-772-52 | |
| Clear TCPS 96-well plate | Fisher Scientific | 08-772-2C | |
| Clear TCPS 48-well plate | Fisher Scientific | 08-772-1C | |
| Cover glasses, circles | Fisher Scientific | 12-545-81 | |
| Falcon tissue culture treated flasks, T25 | Fisher Scientific | 10-126-10 | |
| sticky-Slide 8 Well | Ibidi | 80828 | |
| Superfrost microscope slides | Fisher Scientific | 12-550-15 | |
| Tissue culture treated flasks, T150 | Fisher Scientific | 08-772-48 | |
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Citer Cet Article
McKiel, L. A., Woodhouse, K. A., Fitzpatrick, L. E. A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces. J. Vis. Exp. (155), e60317, doi:10.3791/60317 (2020).