间充质干细胞调节巨噬细胞吞噬作用;定量和成像
Summary
这里提出的方案用于量化和生成间充质干细胞（MSC）介导的巨噬细胞（MΦ）吞噬作用的动态图像，这些聚氧菌（zymosan）颗粒与pH敏感荧光分子偶联。
Abstract
间充质干细胞（MSC）传统上因其再生特性而受到研究，但最近，它们的免疫调节特性一直处于最前沿。它们与免疫细胞相互作用并调节免疫细胞活性。本研究的重点是巨噬细胞吞噬活性的MSC调节。巨噬细胞（MΦ）吞噬作用是先天免疫系统对感染反应的重要组成部分，MSC调节这种反应的机制正在积极研究中。这里介绍的是一种研究与MSC共培养时与pH敏感荧光分子偶联的非调序酶原颗粒的MΦ吞噬作用的方法。随着吞噬活性的增加和标记的酶聚糖颗粒被封闭在吞噬体的酸性环境中，pH敏感分子的荧光强度增加。在适当的激发和发射波长下，使用荧光分光光度计测量吞噬活性，并将动力学数据显示为70分钟内相对荧光单位的变化。为了支持这些定量数据，使用动态成像可视化吞噬细胞活性的变化。使用该方法的结果表明，在共培养中，MSC增强了幼稚和IFN-γ处理的MΦ的非调子化酶的MΦ吞噬作用。这些数据增加了目前对MSC调节先天免疫系统的知识。这种方法可以应用于未来的研究，以充分描绘潜在的细胞和分子机制。
Introduction
间充质干细胞（MSC）是产生结缔组织细胞的祖细胞。MSC存在于成年哺乳动物组织中，可以从骨髓中分离出来1。由于其免疫调节特性，这些细胞被广泛研究2。早期的研究集中在MSC调节T细胞3，4，5，6，但最近，它们对巨噬细胞（MΦ）的调节，巨噬细胞是先天免疫的主要细胞成分，已经受到越来越多的关注7，8，9，10，11，12，13，14.MSC-MΦ相互作用在炎症性疾病治疗中的重要性通过单核细胞/巨噬细胞的消耗在动物模型8中废除MSC的治疗效果这一事实得到了强调。这里的重点是MSC与MΦ的细胞接触相互作用。MSC有能力通过促进从炎症反应到抗炎反应的转变来调节MΦ的表型，导致组织修复活动8，9，10，11，并且已经做了很多工作来证明这些调节机制。在其他情况下，MSC可以支持或加剧MΦ驱动的炎症反应12，13并增强MΦ吞噬活性14，15。然而，严重缺乏现有数据来确定MSC调节MΦ吞噬活性的机制和条件。
MΦ具有识别调理子化（抗体或补体包被）或非调序化病原体导致吞噬作用的受体家族16。后者的激活和活性研究较少17.在非炎症性体外环境中，MSC增强非调子化病原体的MΦ吞噬作用13。然而，在适应性免疫应答期间暴露于淋巴细胞产生的炎症环境后，MΦ对非调序化病原体的识别可能会减少。由自然杀伤细胞和效应淋巴细胞释放的IFN-γ对非调子化颗粒18的MΦ吞噬作用具有抑制作用。建立了共培养模型，研究了MSC直接接触调节MΦ吞噬作用的机制。这里提出的实验目标是确定在MΦ暴露于IFN-γ后，MSC是否调节非调理性病原体的MΦ吞噬作用（图1）。
Protocol
注意：所有培养基制备和细胞培养技术均在无菌条件下使用具有层流流动的生物安全柜进行。所描述的所有培养步骤均使用旨在保持37°C，5%CO2和95%湿度的培养箱进行。 1. 细胞培养 生长培养基的制备 对于 MSC 和 LADMAC，将 50 mL FBS 和 5 mL 100x 抗生素/抗真菌剂混合物加入 500 mL 高糖 DMEM 中。 对于MΦ，加入50 mL FBS，100 mL LADMAC条件培养基（按照第1.2节的?…
Representative Results
在所有时间点计算每组的平均±SEM后，数据以折线图格式显示，Y轴作为相对荧光强度，X轴作为时间。 补充文件1 以电子表格格式提供了来自96孔板动力学读取的原始数据的示例。 在这项研究中，图3A和表3中呈现的最佳结果表明，1）与MSC共培养增强巨噬细胞的吞噬活性，2）IFN-y处理降低巨噬细胞的活性，以及3）与MSC共培养部分?…
Discussion
使用与pH敏感染料偶联的生物颗粒分析吞噬作用是一种相对较新的工具，已被证明比传统的荧光标记颗粒12，19，20具有优势。对于传统的荧光标记颗粒，只有终点分析是可行的。在洗涤或淬灭未被吞噬细胞吸收的颗粒后，使用荧光显微镜和/或光谱荧光测定法进行检测。从光谱荧光测定法获得的定量数据具有检测非吞噬颗粒的潜…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NSF主要研究仪器机制的支持，其授权号为1626093和1919583。
Materials
| 96 Well Black Polystyrene Microplate | MilliporeSigma | CLS3603-48EA | |
| 0.4% trypan blue solution | MilliporeSigma | T8154-20ML | |
| 15 mL and 50 mL Conical Sterile Polypropylene Centrifuge Tubes | ThermoFisher | 339653 | |
| 4-well Chambered Coverglass w/ non-removable wells | ThermoFisher | 155382PK | |
| Antibiotic-Antimycotic (100X) Gibco | ThermoFisher | 15240096 | |
| Axiobserver 7 Imaging System | Zeiss | ||
| Bovine Serum Albumin (BSA) | MilliporeSigma | A8806-1G | |
| Cell lifter | MilliporeSigma | CLS3008-100EA | |
| Culture flasks, tissue culture treated, surface area 75 cm2, canted neck, with cap, filtered | MilliporeSigma | C7231-120EA | |
| D1 ORL UVA [D1] | ATCC | CRL-12424 | Mouse MSC Cell Line |
| DMEM, High Glucose | ThermoFisher | 11965092 | |
| Fetal Bovine Serum, qualified, heat inactivated | ThermoFisher | 16140071 | |
| Hemocytometer | FisherScientific | 02-671-51B | |
| I-11.15 | ATCC | CRL-2470 | Mouse MΦ Cell Line |
| LADMAC Cell Line | ATCC | CRL-2420 | LADMAC cells secrete the growth factor colony stimulating factor 1 (CSF-1). |
| Live-Cell Imaging solution | ThermoFisher | A14291DJ | |
| PBS, pH 7.4 | ThermoFisher | 10010031 | |
| pHrodo Green Zymosan Bioparticles Conjugate | ThermoFisher | P35365 | |
| Recombinant Murine IFN-γ | Preprotech | 315-05 | |
| Spectramax i3X | Molecular Devices | ||
| Sterile Single Use Vacuum Filter Units, 250 mL, 0.2 µm | ThermoFisher | 568-0020 | |
| Sterile syringe filters, 0.2 micrometer | ThermoFisher | 723-2520 | |
| Tissue-culture treated culture dishes, 100 mm x 20 mm | MilliporeSigma | CLS430167-100EA | |
| Trypsin-EDTA (0.05%), phenol red | ThermoFisher | 25300054 |
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Diesen Artikel zitieren
Evans, J. F., Ricigliano, A. E., Morante, A. V., Martinez, E., Vargas, D., Thyagaraj, J. Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging. J. Vis. Exp. (173), e62729, doi:10.3791/62729 (2021).