Summary

分离和新型表面标记组合来标识肝祖亚群富集

Published: February 18, 2017
doi:

Summary

肝损伤都伴随着祖细胞扩张表示异质细胞群。这种蜂窝室的新颖分级允许多个子集的区别。此处所描述的方法示出了流式细胞术分析,并且可用于进一步的测定各种子集的高纯度分离的流动。

Abstract

在慢性肝损伤,祖细胞在称为胆管反应过程中,这也需要炎性细胞浸润和上皮细胞活化的外观扩大。这种炎症反应过程中的祖细胞群体大多被用单表面标志物的调查,无论是组织学分析或通过流式细胞仪为基础的技术。然而,新的表面标志物识别的肝脏祖内的各种不同功能的子集/干细胞室。这里提出的方法描述了分离和详细流式细胞使用新型表面标记组合祖亚群分析。此外,它说明了如何在各种祖细胞亚群可与使用自动磁性高纯度和FACS分选的基础的方法进行分离。重要的是,肝脏的新颖的和简化的酶解允许这些稀有细胞群的具有高可行性的隔离即,相较于其他现有方法优越。这是为进一步研究祖细胞在体外或用于分离高质量的RNA来分析基因表达谱尤其相关。

Introduction

肝再生大多与肝细胞的自我更新的能力相关联。然而,发生与祖细胞活化和扩展,已用其分化成肝细胞和胆管1,2,3,4能力有关的慢性肝损伤。这一点尤其重要,因为,在慢性损伤,肝细胞增殖效果不好。尽管针对祖细胞多基因遗传跟踪研究,他们在肝再生的作用仍然是有争议的5,6,7,8。此外,祖细胞的活化已被链接到在肝脏增加纤维化反应,其损伤9中提出了关于他们的确切作用的问题,10。

祖细胞室的异质性早已被分离出来的使用显微切割或细胞分选基方法1,11表示一个单一的表面标志物前体细胞基因表达研究建议。事实上,最近,一种新的使用表面标志物结合GP38(podoplanin)毫不含糊地挂钩祖细胞的以往单一标记各种子集12。重要的是,这些子集不仅在不同的表面标志物的表达,但伤病期间12还展出了功能改变。

多种动物模型已被用于研究祖细胞的活化和肝再生。似乎各种损伤类型促进不同祖细胞12的子集的激活。这或许可以解释的pH值在人类4观察胆小管反应enotypic分歧。因此,祖细胞的复杂表型和功能分析是枢转,以了解他们在损伤作用和肝脏疾病的导管状反应的真实意义。

除了表面标志的组合,在细胞分离协议的重要区别还基于先前的研究2的结论变得复杂。的研究中的一个显着量寻址祖细胞,在其分离方案有很大的不同( 例如,肝解离(酶组合和处理的持续时间),密度介质,并且离心速度)的作用2。优化的隔离技术,为罕见的细胞群提供更好的生存能力和反射的子集组成,已开发并于最近公布的12。本文的目的是提供一个更DET这种肝细胞分离过程和所述子集分析ailed协议以允许该技术的适当的再现。此外,该协议包括与以前的分离方法来证明相比,新的协议的差异的比较。

Protocol

所有的实验程序,是洪堡大学医学中心的伦理和动物护理委员会批准进行的。 1.材料和缓冲液的制备刚准备用无菌组件和层流罩,以避免细菌污染肝脏消化所需的所有缓冲区。 通过混合49.5毫升RPMI培养基和0.5mL胎牛血清的制备收集缓冲液(CB)(FBS;低内毒素,热灭活的),以达到1%(体积/体积)溶液。存储直到进一步使用冰的解决方案。 注:约合25 CB毫升…

Representative Results

这里介绍的肝脏在含有实质和非实质肝细胞( 图1和图2a)的单细胞悬浮液使用的酶的结果的新的混合物中的消化过程。红血细胞的所述ACK-裂解后,直接流式细胞仪的单细胞悬浮液的分析是可能的( 图1和2)。门控战略涉及的双峰和死细胞( 图2a)排除在外。是阴性CD45,CD31,和ASGPR1细胞被门控。这个群体包?…

Discussion

肝脏炎症和不同来源的损伤触发在于伴随着祖细胞扩增和活化2,3肝再生过程。这些肝祖细胞具有干细胞的特性,并可能发挥各种肝病的病理机制一个显著的作用。

肝祖细胞的异质性早已被建议。使用CD133和GP38的新型表面标记组合可以识别携带不同表面标志的子集,并表示肝损伤12中唯一的一组炎症相关基因的肝脏?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作是由亚历山大·冯·洪堡基金会Sofja Kovalevskaja奖VLK支持。

Materials

RPMI Life Technologies 21875-034
phenol red free DMEM Life Technologies 31053-028
FBS Life Technologies 10270-106
Collagenase P Sigma-Aldrich 11249002001
DNAse-I Sigma-Aldrich 10104159001
Dispase Life Technologies 17105041
ACK Lysing Buffer Life Technologies A10492-01
HBSS Life Technologies 14025-050
PBS Sigma-Aldrich D8537
Sodium Azide Sigma-Aldrich S2002 Prepare 1 % stock solution
10 % BSA Miltenyi Biotec 130-091-376
autoMACS Rinsing Solution Miltenyi Biotec 130-091-222 add 0.5 % (v/v) BSA and store on ice
Phenol-red free DMEM Sigma-Aldrich D1145
counting Beads Count Bright Life Technologies C36950
PI Miltenyi Biotec 130-093-233
FcR Blocking Reagent Miltenyi Biotec 130-092-575
anti-CD31 MicroBeads Miltenyi Biotec 130-097-418
anti-CD45 MicroBeads Miltenyi Biotec 130-052-301
Dead Cell Removal Kit Miltenyi Biotec 130-090-101
anti-Biotin MicroBeads Miltenyi Biotec 130-090-485
CD64 Purified BioLegend 139302 Dilution: 1:100
CD16/32 Purified BioLegend 101302 Dilution: 1:100
CD45 APC/Cy7 BioLegend 103116 Dilution: 1:200, marks hematopoetic cells
CD31 Biotin BioLegend 102504 Dilution: 1:200, marks endothelial cells
ASGPR1 Purified Bio-Techne AF2755-SP Dilution: 1:100, marks hepatocytes
Podoplanin APC BioLegend 127410 Dilution: 1:1400, marks progenior cells
Podoplanin Biotin BioLegend 127404 Dilution: 1:1400
CD133 PE Miltenyi Biotec 130-102-210 use 3 µl, marks progenitor cells
CD133 Biotin BioLegend 141206 Dilution: 1:100
CD34 Biotin eBioScience 13-0341-81 Dilution: 1:100
CD90.2 Pacific Blue BioLegend 140306 Dilution: 1:800
CD157 PE BioLegend 140203 Dilution: 1:600
EpCAM Brilliant Violet 421 BioLegend 118225 Dilution: 1:100
Sca-1 Biotin Miltenyi Biotec 130-101-885 use 10 µl
Mouse IgG2b, κ PE BioLegend 400311
Rat IgG1 PE BioLegend 400407
Rat IgG2b, κ APC/Cy7 BioLegend 400624
Rat IgG2a, κ Biotin BioLegend 400504
Rat IgG2a, κ Brilliant Violet 421 BioLegend 400535
Syrian Hamster IgG APC BioLegend 402012
Syrian Hamster IgG Biotin BioLegend 402004
Normal Goat IgG Control Purified Bio-Techne AB-108-C
Donkey anti-Goat IgG Alexa Fluor 488 Life Technologies A11055 Dilution: 1:800
Streptavidin Alexa Fluor 405 Life Technologies S32351 Dilution: 1:400
100 µm Filter mesh A. Hartenstein PAS3
LS Column Miltenyi Biotec 130-042-401
QuadroMACS separator Miltenyi Biotec 130-090-976
MACSQuant Analyzer 10 Miltenyi Biotec 130-096-343
AutoMACS Pro Separator Miltenyi Biotec 130-092-545
FACS AriaTMIII BD Biosciences
FACSDiva sofware BD Biosciences
Polypropylene Round bottom tube Falcon 352063
Rneasy plus mini kit Qiagen 74134 RLT lysis buffer is included

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Citazione di questo articolo
Julich-Haertel, H., Tiwari, M., Mehrfeld, C., Krause, E., Kornek, M., Lukacs-Kornek, V. Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination. J. Vis. Exp. (120), e55284, doi:10.3791/55284 (2017).

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