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Immunology and Infection

使用的淋巴细胞外渗分析<em>体外</em>人类血脑屏障模型

Published: April 05, 2017
doi:
10.3791/55390
, , , , ,
1Department of Neurology with Institute of Translational Neurology,University Hospital Münster

Summary

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Abstract

淋巴细胞外渗到中枢神经系统（CNS）是免疫监视的关键。淋巴细胞渗出的疾病相关的变化，可能会导致在中枢神经系统的病理生理改变。因此，淋巴细胞迁移的调查中枢神经系统重要的是要了解炎性中枢神经系统疾病和开发新的治疗方法。在这里，我们提出了人类血脑屏障的体外模型来研究淋巴细胞渗出。人脑微血管内皮细胞（HBMEC）的汇合生长在多孔聚对苯二甲酸乙酯的Transwell插入到模拟血脑屏障的内皮。屏障功能是由紧密连接免疫组织化学，跨内皮电阻（TEER）的测量以及伊文思蓝渗透的分析验证。这种模式使罕见的淋巴细胞亚群血细胞渗出的调查，如CD56 亮 CD16 暗淡/ – NK细胞。 Furtherm矿石，其他细胞，细胞因子和趋化因子，疾病相关的改变，以及对淋巴细胞迁移能力不同治疗方案的效果进行研究。最后，炎性刺激对内皮屏障的影响，以及不同的治疗方案可以被分析。

Introduction

从血液进入组织的细胞迁移是免疫监视的关键。特定分子相互作用的序列确保位点特异性外渗到小肠，皮肤，淋巴结，中枢神经系统（CNS），以及其他组织1。在淋巴细胞迁移的改变都参与了许多的广泛传播疾病2的病理生理学。迁移到免疫豁免CNS是被严格调控，因此，该过程的改变参与CNS相关的疾病，如脑脊髓炎3，视神经，中风和多发性硬化（MS）2，4，5，6，7。因此，研究淋巴细胞渗出，以更好地了解疾病的病理生理机制，并制定一个工具，它是非常重要的疾病负担8，9，10，11，12的土壤改良。

淋巴细胞通过不同的路线迁移进入CNS。外渗通过毛细血管小静脉成可经由脉络丛内和跨血脑屏障血-脑脊液屏障蛛网膜下腔已经描述1，13，14，15。跨过血-脑屏障迁移是由淋巴细胞的内皮细胞14的相互作用进行。与此相反，以内皮细胞在外围，CNS的内皮细胞表达大量紧密连接的分子，从而严格限制细胞和蛋白质能够穿过血脑屏障的量小姑娘= “外部参照”> 16。炎症导致紧密连接的松动，并且诱导的粘附分子的表达;因此，增强淋巴细胞迁移进入CNS 1，17，18。

通过血 – 脑屏障外渗是一个多步骤过程。淋巴细胞系链对内皮细胞，然后沿着主要由选择素1，15介导的过程的内皮上滚动。随后，在淋巴细胞上表达由内皮分泌的趋化因子和相应的趋化因子受体之间的相互作用诱导整联的构象变化，从而促进牢固粘附到内皮细胞1。最后，无论是淋巴细胞针对沿着血流内皮屏障抓取transmigrating进入血管周围空间中之前，或立即和直接传动和失速igrate在牢固粘附1，19，20的部位。淋巴细胞外渗所有这些步骤都可以在体外使用不同技术21进行分析。延时录像显微镜用于研究的初步圈养及压延15。粘附分析提供关于牢固停滞的详细信息内皮障碍22。如这里演示轮回测定允许免疫细胞轮回21，23，24，25，26，27，28，29的分析。

使用体外血脑屏障模型的人，我们可以最近表明，较高的MIGRCD56 明亮的 CD16的atory容量暗淡/ – NK细胞相比，他们的CD56 暗淡 CD16 +同行通过在鞘内室21这NK细胞子集的优势体现。因此，我们的实验装置似乎是适用于模拟体内的情况。

Protocol

1.细胞人脑微血管内皮细胞的培养（HBMEC） 细胞培养烧瓶的涂层为了制备纤连蛋白溶液，加入10毫升PBS至15mL离心管中。加入150μL纤维连接蛋白拌匀。 以覆盖所述底部的T-25细胞培养瓶添加2毫升纤连蛋白溶液。在孵化器中孵育的细胞培养瓶为至少3个小时在37℃下。纤连蛋白包被的烧瓶可以在37℃/ 5％CO 2中可以储存2周。 播种和HBMEC的细胞培养从细胞培?…

Representative Results

示出NK细胞和使用人的血脑屏障模型（ 图1A）的T细胞亚群的轮回代表性的结果示。的HBMEC单层的完整性是由紧密连接分子ZO-1，跨内皮电阻（TEER）的测量，和伊文思蓝渗透（ 图1B）的染色验证。以下3 -第4天的培养HBMEC表达的紧密连接分子ZO-1（ 图1B，左）。此外，HBMEC增长在表现出跨内皮电阻（ 图1B中）以及降低的渗透为伊?…

Discussion

在这里，我们提出了一种技术，调查淋巴细胞跨越人类血脑屏障的轮回。 在淋巴细胞迁移至CNS的体外分析是很重要的学习淋巴细胞渗出，潜在的疾病相关的变化，以及新的治疗方法的基本过程。

血 – 脑屏障模型的若干修改是可能的。例如，从上室细胞可以解析，调查了未迁移细胞群的组合物。此外，治疗的HBMEC单层与测定前24小时IFN-γ和TNF-α的可以用来模仿发炎…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study has been supported by the Collaborative Research Centre CRC TR128 “Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease” (Project A9 to H.W. and C.C.G., project B1 to N.S.).

Materials

PBS Gibco 14190-094 without CaCl2 or MgCl2
Fibronectin 1mg/mL Sigma F1141-5MG from bovine plasma
T-25 cell culture flask Greiner BioOne 690160
HBMEC ScienCell 1000
Pelobiotech PB-H-6023
Accutase Sigma A6964-100ML
ECM-b ScienCell 1001-b
FBS ScienCell 1001-b
Penicillin/Streptomycin ScienCell 1001-b
Endothelial cell growth supplement ScienCell 1001-b
Transwell Corning 3472 clear, 6.5mm diameter, 3.0µm pore size
96-well flat bottom plate Corning 3596
Evans blue Sigma E2129-10G stock solution: 1 g/50 mL PBS
B27 Gibco 17504-044 50x concentrated
Infinite M200Pro Tecan
96-well black flat bottom plate Greiner BioOne 675086
48-well plate Corning 3526
RPMI 1640 Gibco 61870-010
Flow Count Fluorospheres Beckman Coulter 7547053
Na-EDTA Sigma E5134
BSA Sigma A2153
Gallios 10-color flow cytometer Beckman Coulter
Kaluza 1.5a Beckman Coulter
TNF-α Peprotech 300-01A
IFN-γ Peprotech 300-02
CD3-PerCP/Cy5.5 Biolegend 300430 clone UCHT1
CD56-PC7 Beckman Coulter A21692 clone N901
CD16-A750 Beckman Coulter A66330 clone 3G8
CD4-FITC Biolegend 300506 clone RPA-T4
CD8-A700 Beckman Coulter A66332 clone B9.11
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Tags

Lymphocyte ExtravasationIn Vitro ModelBlood-brain BarrierInflammatory DiseasesCentral Nervous SystemTransmigration AssayEvans Blue PermeationHBMECFibronectinAccutaseCell Culture Inserts
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Schulte-Mecklenbeck, A., Bhatia, U., Schneider-Hohendorf, T., Schwab, N., Wiendl, H., Gross, C. C. Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier. J. Vis. Exp. (122), e55390, doi:10.3791/55390 (2017).
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