小鼠原发性肝正弦内皮细胞的分离与表征
Summary
在这里，我们概述并演示了原代小鼠肝脏正弦内皮细胞（LSEC）分离方案。该方案基于肝胶原酶灌注、低速离心纯化非实质细胞和CD146磁珠选择。我们还使用流式细胞术和扫描电子显微镜对这些分离的LSECs进行表型和表征。
Abstract
肝正弦内皮细胞（LSECs）是位于循环和肝实质之间界面的特化内皮细胞。LSECs具有独特的形态，其特征在于存在开窗和没有基底膜。LSECs在肝脏的许多病理性疾病中起着至关重要的作用，包括代谢失调，炎症，纤维化，血管生成和癌变。然而，关于LSEC的分离和表征的出版物很少。在这里，该协议讨论了从健康和非酒精性脂肪性肝病（NAFLD）小鼠中分离LSAC。该方案基于小鼠肝脏的胶原酶灌注和磁珠阳性选择非实质细胞以纯化LSECs。本研究通过流式细胞术使用特异性标记物表征LSECs，并通过扫描电子显微镜鉴定特征性表型特征。按照该协议分离的LSECs可用于功能研究，包括粘附和渗透性测定，以及特定目标途径的下游研究。此外，这些LSECs可以合并或单独使用，允许多组学数据生成，包括RNA-seq本体或单细胞，蛋白质组学或磷酸 – 蛋白质组学，以及使用测序（ATAC-seq）进行转座酶可及染色质的测定等。该方案对于研究LSECs与其他肝细胞在健康和疾病中的通讯的研究者非常有用，并允许深入了解LSECs在急性和慢性肝损伤致病机制中的作用。
Introduction
肝正弦内皮细胞（LSECs）排列在肝窦壁上，是肝脏中最丰富的非实质细胞1。LSECs与身体其他毛细血管内皮细胞的区别在于存在开窗和缺乏经典的基底膜或隔膜2，3。因此，LSECs具有独特的表型和结构特征，可增强其通透性和内吞能力，以消除各种循环大分子，包括脂质和脂蛋白。LSECs在实质细胞和非实质细胞（如星状细胞和免疫细胞）之间的串扰中起着关键作用。LSECs通过保持星状细胞和Kupffer细胞处于静止状态来维持肝脏稳态的关键4。LSECs通过介导循环白细胞的粘附和经内皮迁移来调节肝免疫细胞群的组成5，6。在急性和慢性肝损伤7 期间，包括缺血再灌注损伤 （IRI）8、非酒精性脂肪性肝炎 （NASH）9 和肝细胞癌 （HCC），LSECs 会发生表型改变，称为毛细血管形成，其特征为防御和基底膜10 形成。LSECs 的这些表型变化与 LSECs 功能障碍以及获得血栓前、促炎和促纤维化特性有关。
已经开发了几种从小鼠肝脏中分离LSECs的方法11。一些技术依赖于分离非实质和实质细胞，然后进行密度梯度离心以从非实质部分纯化LSECs。该方法的局限性在于LSECs分离的最后步骤中存在污染巨噬细胞，这可能会影响分离的LSECs12的纯度。该方案基于小鼠肝脏的胶原酶灌注和CD146 + 磁珠阳性选择非实质细胞以纯化LSECs。使用这种方法分离的LSECs显示出高纯度和保留的形态和活力。这些 LSEC 是功能研究的最佳选择，包括渗透性和粘附测定，以及目标途径的下游研究。此外，随着人们对在临床研究和发现科学中生成大数据集的兴趣日益浓厚，这些从患有非酒精性脂肪性肝炎（NASH）或其他疾病的健康和患病肝脏中分离出来的高质量LSECs可以合并或单独使用，从而可以生成多组学数据并比较健康与疾病13，14.此外，分离的LSECs可用于开发二维和三维体外模型，如类器官，以破译LSECs中激活的信号通路及其在不同有害刺激下与其他肝细胞的细胞间通讯，并响应各种治疗干预。
Protocol
动物协议是经梅奥诊所的机构动物护理和使用委员会（IACUC）批准的。从杰克逊实验室购买了八周龄的C57BL / 6J雄性小鼠。将小鼠饲养在温度控制的12：12-h明暗循环设施中，并可自由获得饮食。 1.胶原包被培养皿或培养盘的制备 要制成50 mL 0.02 mol/L乙酸，在49.4 mL H2O中加入0.6 mL冰醋酸。 在0.02摩尔/升乙酸中制作50μg/ mL I型胶原蛋白。稀释取决于…
Representative Results
实验示意图及设备设置：在该方案中，使用封闭的灌注回路消化小鼠肝脏，然后通过以50× g 低速离心2分钟分离非实质细胞和肝细胞。使用从非实质部分选择的CD146磁珠分离原代LSECs。实验原理图如图 1A所示。套管通过PV放置，而下腔静脉被绑起来以确保胶原酶通过肝脏的单向灌注（图1B）。内部灌注室配有加热和加湿系统，以确保…
Discussion
在目前的手稿中，我们描述了一种从小鼠肝脏中分离LSEC的方案，包括两步胶原酶灌注和随后的磁活化细胞分选（MACS）。该方案包括以下三个步骤：（1）用无钙缓冲液灌注PV，然后用含胶原酶的缓冲液实现肝细胞分散;（2）低速离心排除肝细胞;和（3）使用抗CD146磁珠从非实质细胞（NPC）中选择性基于MACS的LSECs阳性选择。整个过程可以在3小时内完成。此外，该程序的成本（包括所有耗材）约为每只?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH国家糖尿病，消化和肾脏疾病研究所（1RO1DK122948至SHI）和NIH Silvio O. Conte消化系统疾病研究核心中心P30资助机制（DK084567）的支持。日本科学促进会（JSPS）海外研究奖学金也向钦哲基金会提供了支持。我们还要感谢Gregory J. Gores博士和Steven Bronk博士对胶原酶灌注装置的原创设计和优化。
Materials
| 2.0-inch 20 G Intra Venous (IV) catheter | Terumo, SOmerset, NJ, USA | SR-OX2051CA | |
| 2–3-inch perfuion tray with a hole in the center | customized; made in house | ||
| 405/520 viability dye | Miltenyi, Bergisch Gladbach, Germany | 130-110-205 | |
| 4-inch regular curved dressing forceps | Fisher Brand | FS16-100-110 | |
| 5-0 Perma-Hand silk suture | Ethicon, Raritan, NJ, USA | A182H | |
| Anti-stabilin-2 (Mouse) mAb-Alexa Fluorà 488 | MBL International, Woburn, MA, USA | D317-A48 | |
| BSA stock | Miltenyi, Bergisch Gladbach, Germany | 130-091-376 | |
| Anti-CD146 (LSEC)-PE, anti-mouse | Miltenyi, Bergisch Gladbach, Germany | 130-118-407 | |
| CD146 (LSEC) MicroBeads, mouse | Miltenyi, Bergisch Gladbach, Germany | 130-092-007 | |
| Anti-CD45-Viogreen, anti-mouse | Miltenyi, Bergisch Gladbach, Germany | 130-110-803 | |
| Collagen type I | Corning, Corning, NY, USA | 354236 | |
| Collagenase II | Gibco, Waltham, MA, USA | 17101-015 | |
| Endothelial cells growth medium | ScienCell Research Laboratories, Carlsbad, CA, USA | 211-500 | |
| FcR blocking reagent, mouse | Miltenyi, Bergisch Gladbach, Germany | 130-092-575 | |
| FlowJo software, version 10.6 | Becton, Dickinson and Company | ||
| Hardened Fine scissors | F.S.T, Foster city, CA, USA | 14091-11 | |
| Heated (37 °C) and humidified recirculating perfusion apparatus equipped with Oxygen injection at a rate of 10psi. | customized; made in house | ||
| Hitachi S 4700 scanning electron microscope | Hitachi Inc, Pleasanton, CA, USA | SEM096 | |
| LS columns | Miltenyi, Bergisch Gladbach, Germany | 130-042-401 | |
| MACS pre-separation filters (70 μm) | Miltenyi, Bergisch Gladbach, Germany | 130-095-823 | |
| MACS rinsing buffer | Miltenyi, Bergisch Gladbach, Germany | 130-091-222 | |
| MACS Smart Strainer (70 μm) | Miltenyi, Bergisch Gladbach, Germany | 130-098-462 | |
| MACSQunt flow cytometer | Miltenyi, Bergisch Gladbach, Germany | ||
| Millicell Cell Culture Insert | Millipore Sigma, Burlington, MA, USA | PITP01250 | |
| Nexcelom cell counter | Nexcelom bioscience, Lawrence, MA, USA | Cellometer Auto T4 Plus | |
| Percoll | GE Healthcare, Chicago, IL, USA | 17-0891-01 | |
| Surgical scissors | F.S.T, Foster city, CA, USA | 14001-12 | |
| Very small curved dressing forceps | F.S.T, Foster city, CA, USA | 11063-07 |
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Citazione di questo articolo
Guo, Q., Furuta, K., Aly, A., Ibrahim, S. H. Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells. J. Vis. Exp. (178), e63062, doi:10.3791/63062 (2021).