Anoxia and glucose supplementation preserve neutrophil viability and function
Anoxia and glucose supplementation preserve neutrophil viability and function
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DOI：
10.1182/blood-2015-11-680918
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发表时间：
2016-08-18
期刊：
影响因子：
20.3
通讯作者：
Marteyn, Benoit S.
中科院分区：
文献类型：
--
作者：
Monceaux, Valerie;Chiche-Lapierre, Clarisse;Marteyn, Benoit S.
人类中性粒细胞的功能研究和临床输血由于其分离后寿命短而受到阻碍。在这里，我们证明了中性粒细胞在37℃的缺氧条件下，在3mM葡萄糖和32 μ g/mL二甲基氧基甘氨酸的补充下维持20小时的活力，证明了Mcl-1、增殖细胞核抗原（PCNA）和前caspase-3的稳定。值得注意的是，中性粒细胞的形态（细胞核形状和细胞表面标记物）和功能（吞噬、脱颗粒、钙释放、趋化性和活性氧产生）与血液循环中性粒细胞相当。观察到的中性粒细胞活力的扩展在暴露于氧气后被逆转。延长中性粒细胞寿命可以有效转染质粒（转染效率为40%）和短干扰RNA（白细胞介素-8、PCNA和Bax），作为对中性粒细胞有效和功能性遗传操作的验证，无论是在体外还是体内。在体内，在中性粒细胞减少的豚鼠模型中，输注条件中性粒细胞增加了结肠感染时对福氏志贺氏菌的细菌清除率，强烈表明这些条件中性粒细胞可能适合输注。总之，这种体外调节中性粒细胞应该促进它们的研究，并为基因操作和治疗应用提供新的机会。
Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours inculture media at 37 degrees C under anoxic conditions with 3mM glucose and 32 mu g/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.