Summary

从小鼠组织在母胎界面分离白细胞

Published: May 21, 2015
doi:

Summary

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Abstract

免疫耐受妊娠需要母亲的免疫系统发生,以接受和培养胎儿发育截然不同的变化。性交过程中启动了这种宽容,受精和着床过程中建立和维护整个孕期。母胎耐受性活跃的细胞和分子介质富集在胎儿与母体组织,被称为母胎界面,其中包括胎盘与子宫及蜕膜组织之间的接触部位。这个接口是由间质细胞和浸润白细胞,和它们的丰度和表型特征在怀孕过程中发生变化。白细胞浸润在母胎界面包括嗜中性粒细胞,巨噬细胞,树突细胞,肥大细胞,T细胞,B细胞,NK细胞,和NKT细胞一起创建的局部微环境,维持妊娠。这些细胞或任何inapprop之间的不平衡riate改变其表型被认为是疾病的妊娠的机制。因此,渗透到母胎界面白细胞的研究,以阐明导致妊娠并发症的免疫机制至关重要。本文中所描述的是,采用温和机械解离,随后通过一个健壮酶促解聚用蛋白分解和溶胶原酶鸡尾酒隔离从鼠组织中的白细胞浸润在母胎界面的组合的协议。该协议允许对高数量可行白细胞(> 70%)以足够保守抗原性和功能特性的分离。孤立白细胞然后可通过多种技术,包括免疫,细胞分选,成像,免疫印迹,mRNA的表达,细胞培养,并在体外功能测定法如混合白细胞反应,增殖或细胞毒性测定法来分析。

Introduction

免疫耐受怀孕是一段当母亲的免疫系统内发生截然不同的变化。这些变化让母亲忍受了胎儿,一个半同种异体移植物1。胎儿的父亲表示主要组织相容性复合体(MHC)抗原2,和胎儿细胞已经在母体循环3被发现;然而,胎儿不被拒绝4,5。这个谜尚不完全清楚。

最新的假说认为,母胎耐受性性交过程中创建和受精6,7和维护,以维持一个足月妊娠8-10。这母胎耐受的细目怀孕期间的10-16早期和晚期疾病视为一种机制。母胎容忍包括各种白细胞亚种群,包括T细胞(调节性T细胞,Th1细胞,Th2细胞,和Th17细胞),MAC的参与噬细胞,嗜中性粒细胞,肥大细胞,NK细胞和NKT细胞,树突细胞,和B细胞,在密度和本地化这种变化在整个孕期15,17-19。母胎耐受富集在母胎界面20 -解剖位置在哪里,母亲的免疫系统与胎儿抗原相互作用20,21。

母胎界面胎盘期间,当胎儿绒毛外滋养细胞侵入子宫粘膜22-24创建。在该接口的胎侧,围绕胎儿膜创建一个专门的上皮表面的胎盘内,并且合体细胞控制与母体血液22通过其直接接触的物质交换。在界面的母体侧,蜕膜募集在小鼠占30%至所有蜕膜细胞的50%的白细胞的异质池。除了它们在matern参与人免疫耐受,这些细胞是主要贡献者怀孕期间不同的过程, 例如 ,在保护免受感染,受精生殖道,胚胎着床7.25,蜕膜血管26,血管重构24,27,滋养细胞浸润28,胎盘发展24,25,以及最终,生产和分娩15,17。因此,参与母胎耐受性白细胞的研究是必不可少的阐明了妊娠并发症的发病机制。

虽然使用免疫组织化学和免疫已产生的数据的直接可视化和子宫,蜕膜,胎盘或白细胞29,30的定位,流式细胞仪分析进一步揭示白细胞的特定子集在这些组织中31,32。此外,流式细胞仪已被用来确定密度和脑膜的比例NAL-母胎界面白细胞33和表达水平外和细胞内的蛋白质8-10,34的。流白细胞术分析在母胎界面需要一个单细胞悬液。为了从蜕膜,子宫和胎盘组织中分离白细胞浸润,组织离解的两种方法已被用于:机械和酶。这两种方法都允许渗透白细胞从这些组织的细胞外基质(ECM)的分离。酶组织离解优于机械组织离解,因为它允许更高的产率白细胞较少剪切力相关损害35。因此,机械组织解离需要汇集组织36,其可以增加样品的变异性和非均质性。然而,机械解离可能是选择,当目的抗原可以通过酶解或当改变细胞的功能感兴趣的需要s到被保留( 例如 ,NK细胞的细胞毒性)35。

使用蛋白水解与特定的酶降解的ECM消除了低收率和机械解离观察。一些研究报告用32胰蛋白酶,胶原酶37,DNA31,分散酶38,以及各种酵素32,39的商 ​​业鸡尾酒。然而,性质和不同的酶浓度和消化的持续时间,必须精心定义和验证,以确保维持所需的免疫细胞表面抗原性表位的完整性。各表面结构是由不同的酶差异易受破坏,与某些酶,如胰蛋白酶,是臭名昭著的汽提通过许多单克隆抗体所识别白细胞表面的表位。

这里介绍的是使用proteolyt的方法IC和胶原溶解酶的鸡尾酒,叫的Accutase。此酶溶液是温和足够,而在在母胎界面解离鼠组织仍然有效,并且不需要加入其它离解的试剂或血清以终止分解反应。而且,它已准备好使用所提供,虽然需要解离的时间进行验证,它比上面提到的酶40,41更加健壮。

这两种类型的组织分解的组合的利用率提高了获得的细胞的质量和数量;因此,一些研究已经实施了机械和酶解结合使用,获得满意结果31,32,37。本文所描述的协议建立和验证在我们的实验室;它使用了一个温和的机械解离,随后通过一个健壮的酶分解的组合。该协议允许的分离和进一步研究在白细胞浸润在小鼠组织在母胎界面(子宫,蜕膜,胎盘)。以下协议也保持了细胞表面标记物和产率足够的活细胞用于下游应用程序的完整性就证明了流式细胞术分析。最后,该协议维持细胞制备的分析和构成母胎界面不同小鼠组织的比较一致。

Protocol

与之前在此协议中提到的样品工作,动物伦理委员会批准,必须由当地研究伦理委员会和机构审查委员会给出。当与动物血,细胞,或如本协议的有害因素的工作,适当的生物安全和实验室安全的行动必须遵循的。 1.鼠标操作和组织收集准备一个无菌工作站,并获得无菌工具组织收集。这些工具将包括大型和小型手术剪,钳,细尖镊子。包括标记有相应的组织名称小?…

Representative Results

从母胎界面小鼠组织的解剖示于图1;此过程包括打开腹膜腔( 图1A,B),子宫角( 图1C),包括植入位置( 图1D),和子宫组织的集合( 图1E),胎盘( 图1F)和蜕膜( 图1G),在16.5 DPC。 图2示出了分离的巨噬细胞的形态(F4 / 80 +)的蜕膜和子宫组织在16.5 DPC使用磁性细胞分选收集的。孤立的巨…

Discussion

它记录白细胞浸润的数量和表型特征在母胎界面一致的数据收集是必不可少的理解妊娠并发症的发病机制。几种技术已经描述了促进浸润从在母胎界面的鼠组织白细胞整个怀孕31,38,39,43-46的分离。然而,每种技术不同的是,使用不同的酶或酶组合,需要不同的解离时间,不指定组织的数量时,以及最重要的是,并不总是指定分离的细胞的生存能力。本文所描述的协议允许渗透从鼠组织白细…

Divulgations

The authors have nothing to disclose.

Acknowledgements

NGL是由产妇,围产期和儿童健康的韦恩州立大学围产期倡议的支持。我们非常感谢莫林McGerty和Amy E. Furcron(韦恩州立大学),他们的手稿的读数。

Materials

Magentic Cell Separation
MS Columns
Cell Separator
30μm pre separation filters
Multistand
15mL safe lock conical tubes
MACS Buffer (0.5% bovine serum albumin, 2mM EDTA and 1X PBS)
Reagents
Anti-mouse CD16/CD32
Anti-mouse extracellular antibodies (Table 1)
Sodium azide
Bovine serum albumin (BSA)
LIVE/DEAD viability dye
Fixation buffer solution
FACS Buffer (1% bovine serum albumin, 0.5% sodium azide, and 1X PBS ph 7.2)
Trypan Blue Solution 0.4%
Fetal bovine serum
Additional Instruments
Incubator with shaker
Flow cytometer
Centrifuge
Vacuum system
Incubator
Water bath
Cell counter
Microscope

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Arenas-Hernandez, M., Sanchez-Rodriguez, E. N., Mial, T. N., Robertson, S. A., Gomez-Lopez, N. Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface. J. Vis. Exp. (99), e52866, doi:10.3791/52866 (2015).

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