人脂肪间充质干细胞源凋亡囊泡促进中性粒细胞炎性坏死转变为中性粒细胞凋亡的研究

宫晓静; 祖潇然; 景志强; 张俊雅; 徐亦驰; 韩岩

doi:10.12435/j.issn.2095-5227.25040402

人脂肪间充质干细胞源凋亡囊泡促进中性粒细胞炎性坏死转变为中性粒细胞凋亡的研究

Apoptosis vesicles from human adipose-derived mesenchymal stem cells promote neutrophil apoptosis over inflammatory necrosis

摘要

摘要:
背景间充质干细胞来源凋亡囊泡(mesenchymal stem cells-apoptotic vesicles，MSCs-apoVs)具有减少多形核中性粒细胞(polymorphonuclear neutrophils，PMNs)发生中性粒细胞炎性坏死(neutrophilic inflammatory necrosis，NETosis)，抑制中性粒细胞胞外诱捕网(neutrophil extracellular traps，NETs)生成，促进PMNs凋亡的作用。这使得MSCs-apoVs成为当前治疗NETosis相关免疫疾病的新策略。
目的探讨人脂肪间充质干细胞来源的凋亡囊泡(adipose-derived stem cell-apoptotic vesicles，ADSCs-apoVs)抑制NETosis的机制并促进其凋亡的作用程度。
方法原代分离、培养并鉴定ADSCs，采用高速离心法提取并鉴定由星孢菌素(Staurosporine，STS)诱导凋亡的ADSCs-apoVs。利用差速离心法结合流式无菌分选技术分离提取小鼠骨髓原代PMNs，将PMNs与不同浓度(1 μg/mL、5 μg/mL、10 μg/mL)ADSCs-apoVs共培养8 h，通过免疫荧光及流式检测不同浓度(1 μg/mL、5 μg/mL、10 μg/mL)的ADSCs-apoVs作用后PMNs的NETs生成量，NETosis及凋亡变化。
结果 ADSCs-apoVs通过浓度依赖的双重机制调控PMNs死亡。NETosis抑制显示，5 μg/mL组ADSCs-apoVs与对照组比，能显著抑制H3cit生成量(48.20 ± 0.96 vs 75.30 ± 1.00，P＜0.001)，同时能显著抑制NETosis的发生(NETosis率：36.07% ± 3.07% vs 57.03% ± 5.35%，P=0.002)；10 μg/mL组协同抑制瓜氨酸化组蛋白(citrullinated histone H3，H3cit)生成(24.73 ± 0.90 vs 75.30 ± 1.00，P＜0.001)并降低NETosis率(16.48% ± 8.09% vs 57.03% ± 5.35%，P＜0.001)。凋亡阈值效应显示，10 μg/mL组与对照组相比能显著促进PMNs发生凋亡(50.70% ± 10.86% vs 10.30% ± 1.55%，P=0.001)，并显著促进早期凋亡(14.30% ± 1.97% vs 0.25% ± 0.21%，P＜0.001)及显著促进晚期凋亡(36.40% ± 12.75% vs 10.06% ± 1.40%，P=0.011)，而1、5 μg/mL组ADSCs-apoVs的PMNs凋亡率无显著变化(9.14%±4.82% vs 10.30%±1.55%，P=0.997)，(10.26% ± 7.31% vs 10.30% ± 1.55%，P＞0.999)。
结论 ADSCs-apoVs通过双重途径调控PMNs命运，一是抑制H3cit以抑制NETs的形成进而阻止PMNs发生NETosis；二是激活凋亡通路，促进PMNs大量凋亡。5 μg/mL ADSCs-apoVs是NETosis特异性抑制安全窗浓度，而10 μg/mL ADSCs-apoVs是NETosis抑制协同凋亡激活的有效清除剂量。

Abstract:
Background Mesenchymal stem cell-derived apoptotic vesicles (MSCs-apoVs) have demonstrated the capacity to promote apoptosis in polymorphonuclear neutrophils (PMNs) and reduce their neutrophilic inflammatory necrosis (NETosis). Consequently, MSCs-apoVs have emerged as a novel therapeutic strategy for inhibiting NETs formation by suppressing NETosis in PMNs and enhancing their apoptotic processes.
Objective To investigate the effects of adipose-derived stem cell-derived apoptotic vesicles (ADSCs-apoVs) on promoting PMNs apoptosis and inhibiting NETosis.
Methods Primary mouse bone marrow-derived polymorphonuclear neutrophils (PMNs) were isolated via differential centrifugation combined with sterile fluorescence-activated cell sorting (FACS). PMNs were co-cultured with adipose-derived stem cell apoptotic vesicles (ADSCs-apoVs) at varying concentrations (1, 5, and 10 μg/mL) for 8 hours. NETs formation, NETosis, and apoptosis were quantified using immunofluorescence and flow cytometry.
Results ADSCs-apoVs regulated PMNs fate through dual pathways. NETosis suppression showed that at 5 μg/mL, significant reductions occurred in H3cit production (48.20 ± 0.96 vs 75.30 ± 1.00, P < 0.001), NETosis incidence (36.07% ± 3.07% vs control 57.03% ± 5.35%, P=0.002); At 10 μg/mL, synergistic inhibition was observed for H3cit levels (24.73 ± 0.90 vs 75.30 ± 1.00, P < 0.001), NETosis incidence (16.48% ± 8.09% vs 57.03% ± 5.35%, P < 0.001). Apoptosis threshold effect showed that 10 μg/mL elevated total apoptosis (50.70% ± 10.86% vs 10.30% ± 1.55%, P=0.001), early apoptosis (14.30% ± 1.97% vs 0.25% ± 0.21%, P < 0.001), late apoptosis (36.40% ± 12.75% vs 10.06% ± 1.40%, P=0.011). No significant apoptosis changes occurred at ≤5 μg/mL (10.26% ± 7.31% vs 10.30% ± 1.55%, P > 0.999), 1μg/mL (9.14% ± 4.82% vs 10.30% ± 1.55%, P=0.997).
Conclusion ADSCs-apoVs regulate PMNs fate through dual pathways by inhibiting histone H3 citrullination to suppress NETs formation, thereby preventing NETosis, and concurrently activating apoptosis pathways to induce extensive PMNs apoptosis. 5 μg/mL ADSCs-apoVs defines a therapeutic window for NETosis-selective inhibition, while 10 μg/mL represents an effective clearance dose for synergistic NETosis suppression and apoptosis activation.

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