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Biologie

通过 CLON-G 和 体外 自发死亡测定延长中性粒细胞寿命

Published: May 12, 2023
doi:
10.3791/65132
, , , , , ,
1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College, 2Tianjin Institutes of Health Science, 3Sichuan Provincial People’s Hospital,University of Electronic Science and Technology of China, 4Haihe Laboratory of Cell Ecosystem,Tianjin Medical University, 5Joint Program in Transfusion Medicine, Department of Pathology, Harvard Medical School; Division of Blood Bank, Department of Laboratory Medicine, The Stem Cell Program, Boston Children’s Hospital,Dana-Farber /Harvard Cancer Center

Summary

该协议详细介绍了CLON-G的制备，以将中性粒细胞寿命延长至5天以上，并为使用流式细胞术和共聚焦荧光显微镜评估中性粒细胞死亡提供了可靠的程序。

Abstract

中性粒细胞的平均寿命小于24 h，限制了中性粒细胞的基础研究和中性粒细胞研究的应用。我们之前的研究表明，多种途径可以介导中性粒细胞的自发死亡。通过同时靶向这些途径，半胱天冬酶-溶酶体膜透化-氧化剂-坏死性凋亡抑制加粒细胞集落刺激因子（CLON-G）开发了鸡尾酒，可将中性粒细胞寿命延长至5天以上，而不会显着损害中性粒细胞功能。同时，还制定了用于评估和评估中性粒细胞死亡的可靠且稳定的方案。在这项工作中，我们表明CLON-G可以在 体外 将中性粒细胞寿命延长至5天以上，并且我们展示了FACS和共聚焦荧光显微镜延长中性粒细胞寿命。本报告介绍了CLON-G的制备程序，并展示了中性粒细胞的 体外 自发死亡测定，可用于中性粒细胞的研究和随后的中性粒细胞死亡，从而为中性粒细胞群落提供可靠的资源。

Introduction

已知中性粒细胞包括丰富的细胞质颗粒、烟酰胺腺嘌呤二核苷酸磷酸 （NADPH） 氧化酶、抗菌酶和各种抵御入侵微生物的细胞器;此外，它们是高度运动的，并且是第一个募集到炎症部位的细胞，这意味着中性粒细胞是先天免疫系统的第一道防线1，2。因此，粒细胞输血疗法已成为中性粒细胞减少相关感染的有前途的临床治疗方法，可暂时增强中性粒细胞免疫力3，4，5。最近的发现清楚地表明，中性粒细胞在许多生理病理情况下也起着多面效应器的作用6。中性粒细胞的平均寿命小于24小时，因此，由于与稳定的遗传操作和长期储存相关的限制，中性粒细胞的基础研究和中性粒细胞研究的应用非常困难7，8，9，10，11.有一些细胞系可以部分展示一些中性粒细胞功能，如HL-60，PLB-985，NB4，Kasumi-1和诱导多能干细胞12。这些细胞系可以实现有效的基因编辑和冷冻保存;然而，它们仍然与原发性中性粒细胞有很大不同，因此不能忠实地概括中性粒细胞的功能13。因此，该领域的大多数研究仍然依赖于新鲜分离的原发性中性粒细胞。该领域仍然依赖于生成昂贵且耗时的条件敲除小鼠来研究中性粒细胞中的特定基因功能，但目前还没有人类模型存在。

在努力探索中性粒细胞死亡所涉及的异质过程以及调节这些过程的多种途径14，15之后，最近报道了一种名为CLON-G（半胱天冬酶-溶酶体膜透化-氧化剂-坏死性凋亡抑制加粒细胞集落刺激因子）的新疗法16.CLON-G 由 Q-VD-oph （喹啉基戊酰基-O-甲基天冬氨酸-[-2，6-二氟苯氧基]-甲基酮）、Hsp70（热休克蛋白 70）、DFO（去铁胺）、NAC（N-乙酰半胱氨酸）、Nec-1（坏死抑素-1）和 G-CSF（粒细胞集落刺激因子）组成。中性粒细胞自发性死亡由多种途径介导，包括细胞凋亡、坏死性凋亡和焦亡。Q-VD-oph 通过靶向半胱天冬酶 1、半胱天冬酶 3、半胱天冬酶 8 和半胱天冬酶 917 来抑制中性粒细胞作为泛半胱天冬酶抑制剂的凋亡。中性粒细胞坏死性凋亡依赖于涉及受体相互作用蛋白激酶-1 （RIPK1） 和混合谱系激酶结构域样蛋白 （MLKL） 的信号通路18。作为RIPK1抑制剂，Nec-1抑制中性粒细胞的坏死性凋亡。Hsp70和DFO可抑制溶酶体膜透化（LMP），诱导中性粒细胞凋亡19和焦亡20。活性氧（ROS）通过介导LMP19和细胞凋亡21以及抑制生存信号22，在中性粒细胞死亡中起重要作用。作为一种可以减少ROS积累的抗氧化剂，NAC延缓中性粒细胞死亡。作为一种生长因子，G-CSF激活中性粒细胞存活信号并抑制钙蛋白酶诱导的细胞凋亡23，24。通过同时靶向多个中性粒细胞自发死亡途径，中性粒细胞寿命可以有效地延长到5天以上，而不会影响其功能。CLON-G治疗扩大了中性粒细胞保存、运输和基因操作的可能性，可以加速中性粒细胞群落的研究。同时，基于中性粒细胞死亡的知识，目前批准的细胞死亡测定方案可能会对中性粒细胞14造成意外损害，因此这些方案已被改进为更适合中性粒细胞研究。本报告提供了使用 CLON-G 培养中性粒细胞的详细方案，以及使用流式细胞术和荧光成像对小鼠中性粒细胞进行体外细胞死亡测定。CLON-G对小鼠和人中性粒细胞均有效;但是，此处演示了鼠标示例以简化此协议。NAC的浓度对于小鼠中性粒细胞为1mM，对于人中性粒细胞为10μM。Hsp70具有物种特异性，因此应根据中性粒细胞的来源使用。对于该方案，中性粒细胞是否从外周血或骨髓中分离以及如何分离并不重要。

在本研究中，从小鼠骨髓中分离中性粒细胞以获得足够的中性粒细胞用于实验，因为可以从骨髓中获得约1 x 10 7-1.5 x 10 7中性粒细胞，而只能从单个8-12周龄C57BL / 6小鼠（任何性别）的外周血中分离出1 x 106个中性粒细胞。进行梯度离心以避免FACS分选或MACS分选的机械刺激可能造成的损坏和激活。

Protocol

波士顿儿童医院和实验血液学国家重点实验室（SKLEH）动物护理和使用委员会批准并监督了所有程序。 图1 描述了用CLON-G培养中性粒细胞和 体外 死亡测定的流程图。 1. 使用克隆-G 延长中性粒细胞寿命 注意：所有提到的操作和材料必须是无菌的。确保所有溶液充分混合并均匀分布。 保存 CLON-G 组件通…

Representative Results

CLON-G处理的中性粒细胞的Wright-Giemsa染色形态（图2A-D）和FACS表型（图2E-J）不受影响。基于流式细胞术分析（图3）和荧光图像测定（图4），CLON-G处理的中性粒细胞在24小时内的活力约为90%+。存活率较低可能是由于储存不当、CLON-G 组分浓度不当或起始分离中?…

Discussion

中性粒细胞在先天性和适应性免疫中起着至关重要的作用，它们的稳态受到严格调节。中性粒细胞是人外周血中最丰富的白细胞，它们具有强大而快速的周转。一个健康的成年人每天可以从骨髓中释放1 x 109个中性粒细胞/kg28。因此，中性粒细胞的死亡已成为该领域令人费解的谜团之一，并且已经投入了很多努力来更好地理解它们。半胱天冬酶29，<sup clas…

Divulgations

The authors have nothing to disclose.

Acknowledgements

本项目得到了细胞生态系统创新基金海河实验室（22HHXBSS00036，22HHXBSS00019）、中国医学科学院医学科学创新基金（2021-I2M-1-040，2022-I2M-JB-015）、北京协和医学院中央高校专项研究基金（3332022062）和四川省科技支撑计划（编号：2021YJ0480）的支持。

Materials

0.2 µm syringe filter Pall Corporation 4612 Filtrate prepared CLON-G components.
1.5 mL micro centrifuge tube LABSELECT MCT-001-150 Lab consumable.
15 mL Centrifuge Tubes LABSELECT CT-002-15 Lab consumable.
24 well cell culture plate Falcon 351147 Neutrophil culture plate.
50 mL Centrifuge Tubes LABSELECT CT-002-50 Lab consumable.
BD LSRII BD Instrument for flow cytometry analysis of neutrophil death.
Calcium chloride (CaCl2) Sigma Aldrich C4901 Assitant of Annexin-V binding  to phosphatidylserine.
Confocal microscope Perkinelmer UltraVIEW VOX Instrument for fluorescent analysis of neutrophil death.
Confocal plate NEST 801001-20mm Lab consumable for fluorescent image assay.
Counting beads Thermo Fisher C36950 Quantification in flow cytometry analysis of neutrophil death.
DFO Sigma Aldrich D9533 Component of CLON-G. LMP inhibitor.
Dimethyl sulfoxide ( DMSO) Sigma Aldrich D2650 Solvent for Q-VD-oph and Nec-1s.
Fetal Bovine Serum Gibco 10099141C Component of neutrophil culture basic medium. Nutrition supply.
FITC-Annexin-V BD 51-65874X Annexin-V can bind to phosphatidylserine of aged cells.This is at FITC channel.
Hsp70 Abcam ab113187 Component of CLON-G. LMP inhibitor.
NAC Sigma Aldrich A9165 Component of CLON-G. Antioxidant.
Nec-1s EMD Millipore 852391-15-2 Component of CLON-G. Necroptosis inhibitor.
Penicillin-Streptomycin Solution (PS) Gibco 15070063 Component of neutrophil culture basic medium. Antibiotics to protect cells from bacteria comtamination.
Propidium Iodide (PI) BioLegend 421301 For neutrophil death assay. A small fluorescent molecule that binds to DNA  but cannot passively traverse into cells that possess an intact plasma membrane.
Q-VD-oph Selleck chem S7311 Component of CLON-G. Pan-caspase inhibitor.
Recombinant Human Granulocyte Colony-stimulating Factor for Injection (CHO cell)(G-CSF) Chugai Pharma China GRANOCYTE Component of CLON-G.  Promote neutrophil survival through Akt pathway.
Round-Bottom Polystyrene Tubes Falcon 100-0102 Lab consumable for flow cytometry analysis.
RPMI1640 Gibco C11875500BT Component of neutrophil culture basic medium.
Saline LEAGENE R00641 Solution for flow cytometry analysis of neutrophil death.
Sodium hydroxide (NaOH) FENG CHUAN 13-011-00029 pH adjustion for NAC.
Wright-Giemsa Stain Solution Solarbio G1020 Neutrophil cytospin staining.
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Tags

Neutrophil LifespanCLON-GIn Vitro Spontaneous Death AssayNeutrophil PreservationNeutrophil TransportationNeutrophil Gene ManipulationFlow CytometryFluorescent ImagingCell Culture
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Fan, Y., Teng, Y., Liu, F. t., Ma, F., Hsu, A. Y., Feng, S., Luo, H. R. Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay. J. Vis. Exp. (195), e65132, doi:10.3791/65132 (2023).
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