He Li, Pan Xiaohui, Zhang Xiaoxin, Zhu Guonian, Liu Ruiqi, Qin Chaoyi
Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu, China.
Department of Endocrinology and Metabolism, West China Hospital of Sichuan University, Chengdu, China.
Ann Transl Med. 2022 Aug;10(15):823. doi: 10.21037/atm-22-2890.
Neutrophil extracellular traps (NETs) are a network structure with DNA as skeleton scaffolding a wide range of antimicrobial proteins, which have been shown to contribute to the pathogenesis, persistence, and progression of many chronic inflammatory diseases. The method for isolation of human or mouse NETs has been well-established. However, in some diseases such as atrial fibrillation, the model can only be established in rats. While most related studies isolate rat neutrophils from the peripheral blood, which is insufficient for further acquisition of NETs. Despite the consumptiveness of rat peripheral blood neutrophil isolation, bone-marrow deprived neutrophil is rarely employed and has not been compared to peripheral neutrophil with its NETs secreting capability.
In the current study, a different bone-marrow-oriented strategy was described and conducted. Based on centrifugal program settings, a one-step method and a two-step method was developed and compared. The purity of the isolated neutrophils was determined by Wright's staining and the viability was detected by flow cytometry. NETosis is the specialized cell death of neutrophils accompanied with NETs formation and its degree of rat neutrophils was analyzed by phase contrast microscopy, fluorescence microscopy, and Celigo whole view analysis. The Picogreen dsDNA Assay Kit was used to determine the concentration of NETs obtained from the NET-rich supernatants. The levels of secreted NETs in rat peripheral blood and bone marrow were compared.
Approximately 0.5×10-1×10 neutrophils could be obtained from the bone marrow of a single rat, with viability above 90%, which was comparable to that of neutrophils isolated from humans and mice. The final concentration of NETs obtained from the supernatant ranged from 8-12 µg/mL. The secretion of NETs from bone marrow-derived neutrophils showed a similar trend to polymorphonuclear (PMN) leukocytes. In addition, the extrusion of the intracellular matrix was incomplete during NETosis, and rat NETs showed weak cross-linking capability for forming large-scale net-like structures.
The bone marrow-oriented strategy for isolating rat neutrophils is accessible and repeatable. NETs formation capability is similar between rat bone-marrow and peripheral blood neutrophils.
中性粒细胞胞外陷阱（NETs）是以DNA为骨架、支撑多种抗菌蛋白的网络结构，已证明其在许多慢性炎症性疾病的发病机制、持续存在及进展中发挥作用。人或小鼠NETs的分离方法已成熟。然而，在某些疾病如心房颤动中，模型只能在大鼠中建立。虽然大多数相关研究从外周血中分离大鼠中性粒细胞，但这对于进一步获取NETs是不够的。尽管大鼠外周血中性粒细胞分离消耗较大，但骨髓来源的中性粒细胞很少被采用，且尚未与其分泌NETs的能力与外周中性粒细胞进行比较。
在本研究中，描述并实施了一种不同的以骨髓为导向的策略。基于离心程序设置，开发并比较了一步法和两步法。通过瑞氏染色确定分离的中性粒细胞纯度，通过流式细胞术检测活力。NETosis是中性粒细胞伴随NETs形成的特殊细胞死亡，通过相差显微镜、荧光显微镜和Celigo全景分析来分析大鼠中性粒细胞的NETosis程度。使用Picogreen双链DNA检测试剂盒测定富含NETs的上清液中NETs的浓度。比较大鼠外周血和骨髓中分泌的NETs水平。
从一只大鼠的骨髓中可获得约0.5×10⁶-1×10⁶个中性粒细胞，活力高于90%，这与从人和小鼠中分离的中性粒细胞相当。从上清液中获得的NETs的最终浓度范围为8-12μg/mL。骨髓来源的中性粒细胞分泌NETs的趋势与多形核（PMN）白细胞相似。此外，在NETosis过程中细胞内基质的挤出不完全，大鼠NETs形成大规模网状结构的交联能力较弱。
以骨髓为导向的大鼠中性粒细胞分离策略是可行且可重复的。大鼠骨髓和外周血中性粒细胞之间的NETs形成能力相似。
