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Abstract
Introduction
Protocol
Results
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생체공학

在一个三维胶原蛋白矩阵分析细胞迁移

Published: October 05, 2014
doi:
10.3791/51963
, , , ,
1Institute of Immunology & Experimental Oncology, Center for Biomedical Education and Research (ZBAF),Witten/Herdecke University

Summary

细胞迁移是涉及过多的生理的是，如伤口愈合和免疫反应，和病理生理过程，如癌症的生物现象。在3D胶原基质迁移试验是一种多用途的工具内的三维生理状环境分析不同类型的细胞的迁移特性。

Abstract

迁移的能力，是各种细胞类型的标志，并在一些生理过程，包括胚胎发育，伤口愈合，免疫反应起着至关重要的作用。然而，细胞迁移也是使这些肿瘤细胞从原发肿瘤分离开始转移性扩散的关键机制在癌症。在过去几年内各种细胞迁移测定已开发来分析不同类型的细胞的迁移行为。因为细胞的运动器官的行为明显的二维（2D）之间的不同和三维（3D）环境中，它可以假设该被嵌入在三维环境中细胞迁移的分析将产生更显著细胞迁移数据。所描述的三维胶原蛋白基质迁移测定的优点是，细胞包埋于胶原纤维代表的细胞外基质的主要成分的一种生理三维网络。由于时间推移的视频显微镜真实细胞迁移测定，允许响应于亲洄游因子或酶抑制剂的几种迁移参数以及它们的变化的判断。各种细胞类型可以使用这种技术，包括淋巴细胞/白细胞，神经干细胞，和肿瘤细胞进行分析。同样地，还细胞簇或球状体可以在胶原基质伴随单个细胞从细胞群/球体进入胶原晶格移民的分析中被嵌入。我们的结论是3D胶原基质迁移实验是一种通用的方法来分析在一个生理的3D环境中细胞的迁移。

Introduction

如细胞融合（查看概述见1,2）细胞迁移是涉及过多的生理过程，包括胚胎发育，伤口愈合和免疫反应（综述见3）的另一生物学现象。然而，迁移的能力也是一个前提肿瘤细胞转移（综述见3,4）。

细胞迁移是一个复杂的，而不是由多个信号转导途径的相互作用的各种配体（ 例如 ，细胞因子，趋化因子，生长因子，激素，细胞外基质组分）的受体（ 例如 ，受体酪氨酸激酶的启动指示尚未完全理解的过程，趋化因子受体，整合素）相互作用5，最终导致肌动蛋白细胞骨架随之而来的重组与设计器和黏着物重组和整合素介导的信号6。

<p class ="“jove_content”">为了分析细胞迁移的几种体外和体内细胞迁移测定已经开发了在过去的几十年，包括Boyden小室/ Transwell小室测定法7，划痕试验/伤口愈合测定8-10，三维（ 3D）胶原基质迁移实验11以及活体成像/镜（综述见12）。这些细胞迁移实验都有优点和缺点， 例如 ，关于成本和需求的设备，处理或获得的数据的可靠性。

两个Boyden小室/ Transwell小试验和划痕试验/伤口愈合测定法是容易的，低成本的和发达的测定法来测量细胞迁移的体外 7-10。在Boyden小室/ Transwell小室测定法的细胞接种在含插入孔（约8微米的直径）的顶部-即所谓的上隔室7。可选的，该插入件可以涂覆有细胞外米atrix的组件， 例如 ，纤连蛋白，胶原蛋白等 ，以模仿多个生理环境。同样，内皮细胞可以生长在刀片的顶端，从而模拟内皮细胞屏障13。在定义的时间间隔到下隔室窝藏媒体和补充剂，如生长因子和趋化因子的过程中已通过的孔的那些细胞，被用作读出，以定量细胞迁移（或外渗）。

在划痕试验/伤口愈合测定细胞接种在平板中，并生长至融合10。在实验设置板的依赖性可以预先涂覆有细胞外基质成分，例如纤维连接蛋白。创建一个从无到有/伤口从划痕的每一侧刮细胞单层单细胞后/伤口可以迁移到了国内空白，从而填补/修复它10。的刮伤/伤口的两侧之间的距离被确定中的时间依赖性和作为读出的单元10的迁移活性。然而，细胞增殖之间（这也可能导致划痕/伤口填充/愈合）和细胞迁移的鉴别时，建议在测定随时间推移的视频显微镜和单细胞追踪10结合。

然而，无论是Boyden小室/ Transwell小室法和划痕法/伤口愈合实验，是相当不完善有关生理状细胞环境。在Boyden小室/ Transwell小室测定细胞具有迁移穿过塑料的孔，而在划痕试验/伤口愈合测定细胞接种于一个二维的预包被的塑料板。同样地，它是公认的迁徙行为明显不同的二维和三维环境3之间。例如，成纤维细胞的三维矩阵​​粘连从特征上两个焦点和纤维状粘连不同维基质中α5β1和素αvβ3整合素，桩蛋白，其它细胞骨架成分，和粘着斑激酶14的酪氨酸磷酸化的内容。同样，电池嵌入在3D环境中也显示改变的迁徙行为15。因此，为了更准确地分析细胞迁移的迁移测定法，建议允许测量中的三维生理或生理状环境单细胞的迁移。

活体成像/显微镜是黄金标准的3D生理范围内测量细胞的迁移。这并不只属于胞外基质-细胞相互作用，而且还对不同类型的细胞，如淋巴结17内外渗16或淋巴细胞贩卖期间肿瘤细胞和内皮细胞之间的相互作用，迄今为止，是可能的，因为改进的荧光显微镜技术，例如双光子共聚焦激光扫描显微镜，使用重要的荧光染料和转基因小鼠品系表达荧光蛋白的衍生物12,16,17。此外，活体成像/显微镜可以与手动和自动跟踪电池18的总和。由于2光子共聚焦激光扫描显微镜的需要以及动物（和相应的转基因动物模型）然而，活体成像/显微镜是一种相当高成本的技术。

克服了boyden小室/ Transwell小室试验和划痕试验/伤口愈合测定法的局限性和分析中的三维胶原蛋白基质迁移实验被开发11,19 3D环境中不同细胞类型的迁移。由此，迁移的细胞被嵌入在三维胶原纤维网络，其中更酷似到体内的情况。共同地，由于时间推移的视频显微镜真实细胞迁移测定允许determi国家的几迁移参数以及其响应于亲洄游因子或抑制剂的改变。各种细胞类型可以使用这种技术，包括淋巴细胞和白细胞11,20，造血干/祖细胞21-24，和肿瘤细胞5,25-29进行分析。除了 ​​单个细胞也将细胞簇或球状体，可以嵌入在胶原基质伴随单个细胞从细胞团/球体进入胶原移民的分析中晶格30,31。

该协议提供了关于一个简单但功能强大的技术分析中的3D环境的不同类型细胞的迁徙行为的概述- 体外方法产生的结果是接近的体内情况。

Protocol

1，准备迁移商会制备石蜡/凡士林（1:1）的混合和加热，直到混合物熔化。使用油漆刷和绘制2-3层的石蜡/凡士林（1：1）在玻璃载片的中间混合，根据图1B-1D。 注意：我们使用的是普通玻璃载玻片（76 * 26 * 1.0-1.5毫米（W / D / H）） 迅速涂抹于载玻片上融化的石蜡/凡士林混合，以避免固化而绘制。确保石蜡/石油冻层的长度为约2-2.5厘米和0.3〜0.5厘米的宽度。石蜡/石?…

Representative Results

所使用的三维胶原蛋白基质迁移测定结合的时间推移的视频显微镜和计算机辅助细胞跟踪允许对各种细胞的迁移参数包括基于人群的参数（ 例如 ，平均运动器官的活性）和单细胞为基础的参数的确定（ 例如 ，主动运动，速度，距离时间迁移）。所获得的细胞追踪数据集，数据处理和数据显示的一个例子示于图2中 ，一个小区的跟踪数据文件类似的表（ 图2A）。</s…

Discussion

迁移的能力，是肿瘤细胞4的标志。不脱离原发肿瘤分离并通过周围的结缔组织肿瘤细胞迁移将不能播种继发性病变，这是几乎所有的癌症患者死亡的主要原因的能力。因为这种关系的许多研究都集中于肿瘤细胞的迁移。这些研究的目的是新颖的目标分子和靶通路，有效地阻断肿瘤细胞的迁移，从而损害或减慢转移形成的识别。对于这样的努力可能是β-阻断剂，它已显示出抑制转移性乳腺?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Fritz-Bender-Foundation, Munich, Germany

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Leica DM IL inverted microscope Leica, Wetzlar, Germany
Microscope stage heater Distelkamp Electronic, Kaiserslautern, Germany
JVC C1431 video camera JVC, Bad Vilbel, Germany
Axis 241Q video server Axis communication GmbH, Ismaning, Germany
Mac G5 Computer Apple Macintosh
iMac Apple Macintosh
FileMaker Pro FileMaker GmbH, Unterschleißheim, Germany
Multi-camera video surveillance software(Security Spy) Bensoftware, London, UK
Runtime Revolution Media 2.9.0 RunRev Ltd., Edinburgh, UK
Paraffin Applichem GmbH, Darmstadt, Germany A4264
Petrolatum jelly local drug store
Purecol (liquid collagen) Nutacon BV, Leimuiden, The Netherlands contains 2.9-3.3 mg/ml bovine collagen (95% collagen type I, 5% collagen type IV)
10x MEM Sigma Aldrich, Taufkirchen, Germany M0275
7.5% Sodium Bicarbonate solution Sigma Aldrich, Taufkirchen, Germany S8761
EGF Sigma Aldrich, Taufkirchen, Germany E9644
U73122 Merck Millipore, Darmstadt, Germany 662035 dissolve first in CHCL3; reconstitute in DMSO just prior to use
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Tags

Keywords: Cell Migration3D Collagen MatrixExtracellular MatrixCell LocomotionTime-lapse Video MicroscopyCell TypesCell ClustersSpheroidsMetastasisWound HealingImmune ResponseEmbryonic Development
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Rommerswinkel, N., Niggemann, B., Keil, S., Zänker, K. S., Dittmar, T. Analysis of Cell Migration within a Three-dimensional Collagen Matrix. J. Vis. Exp. (92), e51963, doi:10.3791/51963 (2014).
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