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Introduction
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Imunologia e Infecção

使用"桅杆细胞敲击"小鼠分析体内桅杆细胞的功能

Published: May 27, 2015
doi:
10.3791/52753
, , , , ,
1Department of Pathology,Stanford University School of Medicine, 2Department of Microbiology & Immunology,Stanford University School of Medicine

Summary

我们描述了体 外 衍生桅杆细胞的生成方法，它们被移植到乳腺细胞缺乏的小鼠中，以及不同解剖部位对网状桅杆细胞的表型、数量和分布的分析。此协议可用于评估桅杆细胞 在体内的功能。

Abstract

乳腺细胞 （MCs） 是存在于各种组织的造血细胞，在暴露于外部环境（如皮肤、气道和胃肠道）的部位尤为丰富。以在IgE依赖过敏反应中的有害作用而闻名的MC也成为主机防御毒液和入侵细菌和寄生虫的重要参与者。MC表型和功能可能受微环境因素的影响，微环境因素可能因解剖位置和/或根据免疫反应的类型或阶段而异。因此，我们和其他人 倾向于在体内 方法而不是 体外方法， 以获得对MC函数的洞察。在这里，我们描述了小鼠骨髓衍生培养MC（BMCMCs）的生成方法，它们被收养转移到基因缺MC的小鼠，以及不同解剖部位对收养转移的MC的数量和分布的分析。这种方法，被称为”桅杆细胞敲击” 的方法，已经广泛使用在过去30年评估MC和MC衍生产品的 功能在体内。我们根据近年来开发的替代方法讨论了这种方法的优点和局限性。

Introduction

乳腺细胞（MCs）是由多能骨髓祖先1-3产生的造血细胞。骨髓外泄后，MCs祖先迁移到各种组织，在当地生长因子1-3的影响下发育成成熟的MC。组织驻地MC战略性地位于主机环境界面，如皮肤、气道和胃肠道，它们作为抵御外部侮辱的第一道防线。MCs 通常根据其”基线”表型特征及其解剖位置进行子分类。在小鼠中，有两种类型的MC被描述：”结缔组织型”MC（CTMC）和粘体MC（MMCs）1-3，7，8。CTMC通常位于静脉注射器和神经纤维附近，并居住在血清腔中，而MMC在肠道和呼吸粘膜1-3中占据腹腔内位置。

研究9-13年MC的生物功能，已应用多种方法。许多小组都专注于使用细胞系的体外方法（如人类MC线HMC114或LAD215，16）、体外衍生MC（如人类外周血源性MC17，或小鼠骨髓衍生） 培养的MC [BMCMCs]18，胎儿皮肤衍生MC [FSCMCs]19和腹腔细胞衍生MC [PCMC]20）或来自不同解剖站点的前体内分离的MC。所有这些模型被广泛用于研究MC生物学的分子细节，例如MC激活中涉及的信号通路。然而，MCs生物学的一个重要方面是，它们的表型和功能特征（如细胞质颗粒蛋白酶含量或对不同刺激的反应）可以通过解剖位置和微环境2，7进行调节。由于在体内遇到的这些因素的确切混合物可能很难在体外繁殖，我们赞成使用体内方法来深入了解MC功能9。

存在一些具有遗传 MC 缺陷的小鼠菌株，例如广泛使用的 WBB6F1–套件W/W-v或 C57BL/6-套件W-sh/W-sh小鼠。这些小鼠缺乏KIT（CD117）的表达和/或活性，KIT是主要MC生长因子干细胞因子（SCF）21，22的受体。因此， 这些小鼠有一个深刻的MC缺陷，但也有额外的表型异常有关他们的c-套件突变（在WBB6F1–套件W/W-v小鼠）或大染色体反转的影响，导致减少c-套件表达（在C57BL/6-套件W-sh/W-sh小鼠）9，10，12，23。最近，一些具有c-套件独立构成MC缺乏症的小鼠株被报告为24-26。所有这些小鼠和一些额外的新型诱导MC缺陷小鼠最近已详细审查9，10，13。

在这里，我们描述了小鼠骨髓衍生培养MC（BMCMCs）的生成方法，它们被收养转移到缺乏MC的老鼠，以及不同解剖部位对收养转移的MC的数量和分布的分析。这种所谓的 “桅杆细胞敲击” 方法可用于评估MC和MC衍生产品 在体内的功能。我们根据近年来开发的替代方法讨论了这种方法的优点和局限性。

Protocol

所有动物护理和实验都是按照国家卫生研究院的指导方针和斯坦福大学动物护理和使用机构委员会的具体批准进行的。 1. 骨髓衍生培养乳腺细胞（BMCMCs）的生成和特征。 注意：捐赠者BMCMC应该来自与接受MC缺乏小鼠具有相同遗传背景的骨髓细胞。雄性供体BMCMC不适合雌性小鼠的移植。女性衍生的捐赠者BMCMC将成功地融入男性和女性接受者。 骨髓提?…

Representative Results

图1中概述了”桅杆细胞敲击”方法，包括BMCMCs的生成、应移植到缺MC小鼠体内的细胞数量（如果根据实验设计指示，数量可以有所变化）和根据注射部位的增殖和实验之间的间隔（如果指示，此间隔也可能有所不同：例如，MC细胞质颗粒中存储的调解员的内容随着时间的增加而稳步增加。图2显示DMEM介质中含有20%WEHI-3细胞条件介质作为IL-3来源的具有代表…

Discussion

在最初的描述38年之后的近30年，”桅杆细胞敲击“方法继续提供有价值的信息，说明MC在体内可以做什么或不能做什么。长期以来，人们一直认为MC的功能仅限于它们在过敏症中的作用。使用”桅杆细胞敲击“方法生成的数据改变了这一观点，通过提供证据，证明MC除了其他功能外，可以在宿主防御某些病原体4，39或毒气28，31方面发挥关键作用，甚至可以抑?…

Declarações

The authors have nothing to disclose.

Acknowledgements

N.G.是法国”法国重建基金会”和菲利普基金会的奖学金获得者;R.S. 由露西尔·帕卡德儿童健康基金会和斯坦福NIH/NCRR CTSA奖号码UL1 RR025744支持;P.S. 得到马克斯·卡德基金会和奥地利科学院的马克斯·卡德研究金和奥地利科学基金（FWF）施罗德研究金的支持：J3399-B21：S.J.G. 承认来自国家卫生研究院的支持，资助 U19 AI104209、NS 080062 和加州大学烟草相关疾病研究计划;L.L.R. 承认关节炎国家研究基金会 （ANRF） 和国家卫生研究院资助 K99AI110645。

Materials

1% Antibiotic-Antimycotic Solution Corning cellgro 30-004-Cl
3 ml Syringe Falcon 309656
35 mm x 10 mm Dish Corning cellgro 430588
5 ml Polystyrene Round Bottom Tube Falcon 352058
Acetic Acid Glacial Fisher Scientific A35-500
Alcian Blue 8GX Rowley Biochemical Danver 33864-99-2
Allegra 6R Centrifuge Beckman
Anti-mouse CD16/32 (clone 93) Purified eBioscience 14-0161-81
2-Mercaptoethanol Sigma Aldrich M7522
BD 1 ml TB Syringe BD Syringe 309659
BD 22G x1 (0.7 mm x 25 mm) Needles BD Precision Glide Needle 205155
BD 25G 5/8 Needles BD Syringe 305122
BD 30G x1/2 Needles BD Precision Glide 305106
Blue MAX Jr, 15 ml Polypropylene Conical Tube Falcon 352097
Chloroform Fisher Scientific C298-500
Cytoseal 60 Mounting Medium Richard-Allan Scientific 8310-4
Cytospin3 Shandon NA
DakoCytomation pen Dako S2002
Dulbecco Modified Eagle Medium (DMEM) 1x Corning cellgro 15-013-CM
Ethanol Sigma Aldrich E 7023-500ml
Fetal Bovine Serum Heat Inactivated Sigma Aldrich F4135-500ml
FITC Conjugated IgG2b K Rat Isotype Control eBioscience 14-4031-82
Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8) eBioscience 11-1171-82
Formaldehyde Fisher Scientific F79-500
Giemsa Stain Modified Sigma Aldrich GS-1L
Isothesia Henry Schein Animal Health 29405
May-Grunwald Stain Sigma Aldrich MG-1L
Multiwell 6 well plates Falcon 35 3046
Olympus BX60 Microscope Olympus NA
Paraplast Plus Tissue Embedding Medium Fisher Brand 23-021-400
PE Conjugated IgG Armenian Hamster Isotype Control eBioscience 12-4888-81
Phosphate-Buffered-Saline (PBS) 1x Corning cellgro 21-040-CV
Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1) eBioscience 12-5898-82
Propidium Iodide Staining Solution eBioscience 00-6990-50
Recombinant Mouse IL-3 Peprotech 213-13
Safranin-o Certified Sigma Aldrich S8884
Tissue culture flasks T25 25 cm2 Beckton Dickinson 353109
Tissue culture flasks T75 75 cm2 Beckton Dickinson 353110
Toluidine Blue 1 % Aqueous LabChem-Inc LC26165-2
Recombinant Mouse SCF Peprotech 250-03
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Tags

Mast CellsIn VivoKnock-in MiceBone Marrow-derived Cultured Mast CellsAdoptive TransferAnatomical DistributionHost DefenseAllergic Reactions
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Gaudenzio, N., Sibilano, R., Starkl, P., Tsai, M., Galli, S. J., Reber, L. L. Analyzing the Functions of Mast Cells In Vivo Using ‘Mast Cell Knock-in‘ Mice. J. Vis. Exp. (99), e52753, doi:10.3791/52753 (2015).
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