全反式维甲酸诱导分化的HL-60在佛波酯刺激下产生中性粒细胞胞外诱捕网的特性  

The characteristics of neutrophil extracellular traps produced by all-trans retinoic acidinduced dHL-60 under PMA stimulation

作　　者：刘望 方金花 洪天添 黄嘉祺 赵柏松 方颖[1] 吴建华[1] 林蒋国 LIU Wang;FANG Jinhua;HONG Tiantian;HUANG Jiaqi;ZHAO Baisong;FANG Ying;WU Jianhua;LIN Jiangguo(School of Biosciences and Bioengineering,South China University of Technology,Guangzhou 510006,P.R.China;Research Center of Medical Sciences,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,P.R.China;Department of Anesthesiology,Guangzhou Women and Children’s Medical Center,Guangzhou 510623,P.R.China;Department of Emergency Medicine,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,P.R.China)

机构地区：[1]华南理工大学生物科学与工程学院,广州510006 [2]广东省人民医院广东省医学科学院医学研究部,广州510080 [3]广州市妇女儿童医疗中心麻醉科,广州510623 [4]广东省人民医院广东省医学科学院急诊科,广州510080

出　　处：《生物医学工程学杂志》2022年第5期909-918,共10页Journal of Biomedical Engineering

基　　金：国家自然科学基金项目(32071303,31771012,12072117,12172137)。

摘　　要：中性粒细胞释放的胞外诱捕网(NETs)是一把双刃剑,了解NETs的形成机制对疾病治疗具有重要意义。然而,寿命短、个体差异大以及无法进行基因编辑使得中性粒细胞难以被用来破译NETs的形成。因此,寻找模式细胞替代中性粒细胞来研究NETs的形成机制是非常有必要的。在本研究中,我们使用不同浓度(0、0.1、1和10μmol/L)的全反式维甲酸(ATRA)对HL-60细胞进行不同天数的分化(1、3、5和7天)。通过检测细胞活力和细胞核形态,我们证实HL-60细胞经ATRA处理5天后可以分化为中性粒细胞样细胞(dHL-60)。使用免疫荧光染色检测NETs的形成,结果表明1μmol/L的ATRA分化5天得到的dHL-60细胞在佛波酯(PMA)刺激下产生的NETs与中性粒细胞产生的相当,且都不伴随组蛋白H3瓜氨酸化。此外,dHL-60细胞形成的NETs依赖于NADPH而不依赖于PAD4,这与中性粒细胞一致。这些结果表明,经1μmol/L ATRA分化5天的dHL-60细胞可作为中性粒细胞的模式细胞,用于NETs形成机制的研究。Extracellular traps released by neutrophils(neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations(0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid(ATRA) to differentiate HL-60 cells for different days(1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells(dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate(PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together,these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.

关 键 词：中性粒细胞胞外诱捕网 HL-60 全反式维甲酸 分化 

分 类 号：R392[医药卫生—免疫学]