JoVE Journal > Immunology and Infection
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Abstract
Introduction
Protocol
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参考文献
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免疫学と感染

毛里小肠内皮淋巴细胞的内质成像

Published: June 24, 2019
doi:
10.3791/59853
,
1Center for Immunity and Inflammation, Department of Pathology, Immunology and Laboratory Medicine,Rutgers New Jersey Medical School

概要

我们描述了一种通过倒转旋转盘共聚焦显微镜,利用鼠小肠的细胞内成像来可视化GFP标记的IELs的方法。该技术能够跟踪粘液中的活细胞长达4小时,并可用于研究各种肠道免疫-皮皮相互作用。

Abstract

表达βT细胞受体(*IEL)的内皮淋巴细胞在肠道上皮的免疫监测中起着关键作用。部分由于缺乏一个明确的配体为β+T细胞受体,我们对α®IEL活化的调节及其在体内的功能的理解仍然有限。这就要求制定替代策略,以询问在调节[IEL功能]时涉及的信号通路,以及这些细胞对局部微环境的响应能力。尽管人们普遍认为® IEL 能够限制病原体的易位,但使用生命内成像对于了解稳态 IEL/上皮相互作用的时空动力学和对侵入性病原体的反应至关重要。在此,我们提出了一种使用倒转旋转盘共聚焦激光显微镜在GFP +T细胞报告小鼠的小肠道粘孔中可视化IEL迁移行为的协议。尽管这种方法的最大成像深度相对于使用双光子激光扫描显微镜有限,但旋转盘共聚焦激光显微镜具有高速图像采集的优势,减少了光漂白和光。使用4D图像分析软件,T细胞监测行为及其与相邻细胞的相互作用可以在实验操作后进行分析,以提供对肠道粘细胞内IEL激活和功能的更多洞察。

Introduction

内皮淋巴细胞(IEL)位于肠道上皮内,在侧细胞间空间1中沿基底膜和相邻上皮细胞之间发现。每5-10个上皮细胞大约有一个IEL;这些IEL作为哨兵,提供免疫监测的肠道上皮屏障2的广大。表达β+T细胞受体(TCR)的IELs占鼠小肠中IEL总种群的60%。对T细胞缺乏小鼠的研究表明,这些细胞对肠道损伤、炎症和感染的反应具有大部分的保护作用。尽管Tcrd敲除小鼠6的产生,我们对IEL生物学的理解仍然有限,部分原因是*TCR所承认的配体尚未确定7。因此,由于缺乏研究该细胞群的工具,因此难以研究βTCR激活和在生理和病理条件下的作用。为了填补这一空白,我们开发了活成像技术,以可视化 IEL 迁移行为和与邻近肠细胞的相互作用,以此提供对 [ IEL 功能和对体内外部刺激的反应) 的更多见解。

在过去十年中,生命内成像显著扩展了我们对肠道生物学多个方面涉及的分子事件的理解,包括上皮细胞脱落8、上皮屏障功能调节9 ,10,骨髓细胞采样的发光含量11,12,和宿主微生物相互作用11,13,14,15,16.在IEL生物学的背景下,使用生命内显微镜揭示了IEL运动的时空动力学和调解其监测行为的因素13,14,15, 16.TcrdH2BeGFP(TcrdEGFP)报告小鼠的发育表明,*IEL在上皮内具有很高的运动性,并且表现出一种对微生物有反应的独特监测行为。感染17,13,14。最近,另一种β细胞报告器小鼠被开发(Tcrd-GDL),在细胞质中表达GFP,允许整个细胞18的可视化。类似的方法也被用来研究特定化学素受体的需求,如G蛋白耦合受体(GPCR)-18和-55,对IEL运动19,20的动态。在没有细胞特异性报告器的情况下,针对CD8+的荧光结合抗体用于可视化和跟踪体内的IEL运动能力19,20。虽然双光子激光扫描显微镜通常用于生命内成像,但使用旋转盘共聚焦激光显微镜具有独特的优势,可以捕获以最小的背景噪声捕获高速和高分辨率多通道图像。该技术非常适合阐明肠道粘液复杂微环境中免疫/表皮相互作用的时空动力学。此外,通过使用各种转基因和/或敲除小鼠模型,这些研究可以深入了解肠道免疫和/或上皮细胞功能的分子调节。

Protocol

所有研究均根据罗格斯新泽西医学院比较医学资源批准的协议,在实验室动物护理评估与认证协会(AALAC)认可的设施中进行。 1. 鼠标准备 注:以下程序,包括动物准备和手术,需要30-40分钟。 手术前,打开显微镜,将显微镜上的封闭培养箱加热至37°C。 在C57BL/6背景上对8-10周大的TcrdEGFP小鼠进行实验,这些实验来自伯纳德·马列森(INSERM,法国巴黎)?…

Representative Results

使用 TcrdEGFP 实验鼠的维生成像技术,我们之前已经表明 , * IELs 表现出动态监视行为, 它们沿着基底膜迁移到侧细胞间空间 (LIS) 中,以稳定的方式巡视上皮状态 (图 2, 影片1。 这种方法还可用于评估特定细胞信号通路和/或受体的抑制如何影响[]IEL迁移行为。例如,白细胞介素(IL)-15是一种多性细胞因子,对ΑIEL平衡症23、24至关重要。</su…

Discussion

生命体间显微镜技术的发展为观察亚细胞结构的重组提供了前所未有的机会, 25和细胞迁移行为13,14,15,16,26在否则无法进入的纸巾。在体内研究IEL的调节和功能?…

開示

The authors have nothing to disclose.

Acknowledgements

这项工作由NIH R21 AI143892、新泽西健康基金会赠款、布施生物医学赠款(KLE)支持。我们感谢马德琳·胡协助编辑手稿并提供代表性结果中显示的数据。

Materials

35mm dish, No. 1.5 Coverslip MatTek P35G-1.5-14-C
Alexa Fluor 633 Hydrazide Invitrogen A30634
BD PrecisionGlide Hypodermic needles – 27g Thermo Fisher Scientific 14-826-48
BD Slip Tip Sterile Syringe – 1 ml Thermo Fisher Scientific 14-823-434
BD Tuberculin Syringe Thermo Fisher Scientific 14-829-9
Dissecting scissors Thermo Fisher Scientific 08-940
Electrocautery Thermo Fisher Scientific 50822501
Enclosed incubation chamber OKOLAB Microscope
Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length Roboz RS-7983-3
Hank's Balanced Salt Solution Sigma-Aldrich 55037C
Hoechst 33342 Invitrogen H3570
Imaris (v. 9.2.1) with Start, Track, XT modules Bitplane Software
Inverted DMi8 Leica Microscope
IQ3 (v. 3.6.3) Andor Software
Ketamine Putney Anesthesia
Kimwipes VWR 21905-026
McPherson-Vannas scissors 3” (7.5 cm) Long 5X0.15mm Straight Sharp Roboz RS-5600
Non-absorbable surgical suture, Silk Spool, Black Braided Fisher Scientific NC0798934
Nugent Forceps 4.25” (11 cm) Long Angled Smooth 1.2mm Tip Roboz RS-5228
Puralube Vet Ointment Dechra Lubricating Eye Ointment
Spinning disk Yokogawa CSU-W1 with a 63x 1.3 N.A. HC PLAN APO glycerol immersion objective, iXon Life 888 EMCCD camera, 405 nm diode laser, 488 nm DPSS laser, 640 nm diode laser Andor Confocal system
Xylazine Akorn Anesthesia
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参考文献

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タグ

Intravital ImagingIntraepithelial LymphocytesMurine Small IntestineMicroscopyImmune Cell InteractionsEpitheliumReal-time ImagingMucosal ImmunityEpithelial BiologyCancer BiologySurgical TechniqueAnesthetized Transgenic MouseEGFPGamma-delta T-cell ReceptorHoechst 33342 DyeAbdominal IncisionMesenteryPeritoneal CavitySuture Placement

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記事を引用
Jia, L., Edelblum, K. L. Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine. J. Vis. Exp. (148), e59853, doi:10.3791/59853 (2019).
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