通过生命内显微镜在斑马鱼胚胎的分孔细菌感染过程中巨噬细胞细胞死亡的可视化
Summary
该协议描述了一种技术，用于可视化在胚胎斑马鱼的巨噬行为和死亡在分枝杆菌感染期间。包括制备细菌、胚胎感染和生命内显微镜的步骤。在涉及感染或无菌炎症的类似情况下，此技术可应用于观察细胞行为和死亡。
Abstract
斑马鱼是研究先天免疫细胞行为的优秀模型生物体，由于其透明性，在早期发育过程中完全依赖于其先天免疫系统。斑马鱼分枝杆菌（M.Marinum）感染模型在研究宿主对分枝杆菌感染的免疫反应方面已经确立。有人建议，不同的巨噬细胞死亡类型将导致分管感染的不同结果。在这里，我们描述了一个协议，使用生命内显微镜来观察斑马鱼胚胎在M.Marinum感染后巨噬细胞死亡。斑马鱼转基因线，专门标记巨噬细胞和嗜中性粒细胞通过肌肉内显微注射荧光标记M.Marinum在中脑或躯干感染。受感染的斑马鱼胚胎随后安装在低熔胶琼脂上，并通过X-Y-Z-T尺寸的共聚焦显微镜观察。由于长期实时成像需要使用低激光功率来避免光漂白和光毒性，因此强烈建议使用强表达转基因。该协议有助于可视化体内的动态过程，包括免疫细胞迁移、宿主病原体相互作用和细胞死亡。
Introduction
分体菌感染已被证明会导致宿主免疫细胞死亡1。例如，衰减菌株会触发巨噬细胞凋亡并控制感染。然而，毒株会触发溶血细胞死亡，导致细菌传播1，2。考虑到这些不同类型的细胞死亡对宿主抗分体细菌反应的影响，需要详细观察体内分管感染期间的巨噬细胞死亡。
测量细胞死亡的常规方法是使用死细胞污渍，如安宁五，TUNEL，或丙氨酸橙/碘化钠染色3，4，5。然而，这些方法无法揭示体内细胞死亡的动态过程。活成像6已经促进了体外细胞死亡的观察。然而，结果是否准确模拟生理状况仍不清楚。
斑马鱼是研究宿主抗分枝杆菌反应的极好模型。它有一个高度保守的免疫系统，类似于人类，一个容易操纵的基因组，和早期胚胎是透明的，允许活成像7，8，9。感染M.Marinum后，成年斑马鱼形成典型的成熟肉芽肿结构，胚胎斑马鱼形成早期肉芽肿等结构9，10。先天免疫细胞-细菌相互作用的动态过程在斑马鱼M.Marinum感染模型11、12中已经探索。然而，由于高时空分辨率要求，有关先天免疫细胞死亡的细节在很大程度上仍未定义。
在这里，我们描述了如何可视化由体内分体细菌感染引发的巨噬细胞细胞死亡过程。此协议也可以应用于可视化发育和炎症期间体内的细胞行为。
Protocol
斑马鱼是在标准条件下养殖的，符合实验室动物动物福利伦理审查准则（GB/T 35823-2018）。本研究的所有斑马鱼实验均获批（2019-A016-01），在复旦大学上海公共卫生临床中心进行。 1. M. 马里南单细胞接种制剂 （图 1） 从 -80°C 解冻 Cerulean-荧光M. 大麻甘油，用 10% （v/v） OADC、0.25% 甘油和 50 μg/mL 湿霉素接种 7H10 琼脂板。在32°C下孵育板?…
Representative Results
分枝杆菌感染可以触发不同的宿主响应，根据感染途径。在此协议中，斑马鱼胚胎通过肌肉内微注射荧光标记的细菌进入中脑或躯干（图3），并通过共聚焦活成像进行观察。通过这两种途径的感染将局部限制感染，导致先天免疫细胞招募和随后的细胞死亡。 可视化先天免疫细胞死亡的细节是具有挑战性的。溶血细胞死亡发生在很短的时间窗口，需要高?…
Discussion
该协议描述了分管感染期间巨噬菌体死亡的可视化。根据细胞膜完整性等因素，感染驱动细胞死亡可分为凋亡和裂解细胞死亡24、25。Lytic细胞死亡对生物体的压力比凋亡更大，因为它触发强烈的炎症反应24，25。 由于要求高时空分辨率、适当的共聚焦显微镜设置和强烈的转基因表达，很难观察体内的溶血细胞?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢温子龙博士分享斑马鱼菌株，斯特凡·奥勒斯博士和大卫·托宾博士分享马兰姆相关资源，何跃鹏协助进行图体准备。这项工作得到了国家自然科学基金（81801977）（B.Y.）、上海市卫生委员会杰出青年培训计划（2018YQ54）（B.Y.）、上海航海项目（18YF1420400）和上海结核病重点实验室开放基金（2018KF02）（B.Y.）的支持。
Materials
| 0.05% Tween-80 | Sigma | P1379 | |
| 10 mL syringe | Solarbio | YA0552 | |
| 10% OADC | BD | 211886 | |
| 3-aminobenzoic acid | Sigma | E10521 | |
| 5 μm filter | Mille X | SLSV025LS | |
| 50 μl/ml hygromycin | Sangon Biotech | A600230 | |
| 7H10 | BD | 262710 | |
| 7H9 | BD | 262310 | |
| A glass bottom 35 mm dish | In Vitro Scientific | D35-10-0-N | |
| Agarose | Sangon Biotech | A60015 | |
| Confocal microscope | Leica | TCS SP5 II | |
| Enviromental Chamber | Pecon | temp control 37-2 digital | |
| Eppendorf microloader | Eppendorf | No.5242956003 | |
| Glass microscope slide | Bioland Scientific LLC | 7105P | |
| Glycerol | Sangon Biotech | A100854 | |
| Incubator | Keelrein | PH-140(A) | |
| M.marinum | ATCC BAA-535 | ||
| Microinjection needle | World Precision Instruments | IB100F-4 | |
| Microinjector | Eppendorf | Femtojet | |
| Micromanipulator | NARISHIGE | MN-151 | |
| msp12:cerulean | Ref.: PMID 25470057; 27760340 | ||
| Phenol red | Sigma | P3532 | |
| PTU | Sigma | P7629 | |
| Single concavity glass microscope slide | Sail Brand | 7103 | |
| Sonicator | SCICNTZ | JY92-IIDN | |
| Spectrophotometer (OD600) | Eppendorf AG | 22331 Hamburg | |
| Stereo Microscope | OLYMPUS | SZX10 | |
| Tg(mfap4:eGFP) | Ref.: PMID 30742890 | ||
| Tg(coro1a:eGFP;lyzDsRed2) | Ref.: PMID 31278008 | ||
| Tg(mpeg1:LRLG;lyz:eGFP) | Ref.: PMID 27424497; 17477879 |
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Citazione di questo articolo
Niu, L., Wang, C., Zhang, K., Kang, M., Liang, R., Zhang, X., Yan, B. Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy. J. Vis. Exp. (143), e60698, doi:10.3791/60698 (2019).