ONO-AE-248诱导的中性粒细胞非凋亡、非坏死性死亡中未出现DNA小片断化的分子机制初探

Study on the molecular mechanism of the lack of the oligonucleosomal DNA fragmentation in the non-apoptotic, non-necrotic neutrophil death induced by ONO-AE-248

目的探讨ONO-AE-248诱导的中性粒细胞非凋亡非坏死性死亡中没有出现DNA小片段的分子机制，为进一步确立这种新型细胞死亡形式提供实验室依据。方法体外新鲜分离人外周血中性粒细胞与ONO—AE-248共同培养，TUNEL法结合激光共聚焦扫描显微镜检测凋亡细胞并观察细胞核形态；Westernblotting检测caspase-3的活性；RT—PCR检测DNA断裂因子（DFF40）的表达水平。实验中同时设立空白组、LPS抑制凋亡组和TNF—α．促进凋亡组为对照。结果ONO—AE-248刺激培养的中性粒细胞经TUNEL法检测未见阳性细胞，细胞核染色密度较低，边界不清，分叶不明显，核形较大；Western blotting结果显示ONO—AE-248对caspase一3前体裂解无显著影响；RT—PCR结果显示ONO—AE-248诱导了DFF40表达明显下调。结论ONO-AE-248诱导的中性粒细胞死亡过程中细胞核DNA断裂方式明显不同于自发性凋亡，DFF40表达下调可能是其没有出现DNA小片段化的主要原因之一。

To investigate the molecular mechanism of the lack of the oligonucleosomal DNA fragmentation in the neutrophil death induced by ONO-AE-248. Methods Human neutrophils were cultured in the presence or absence of ONO-AE-248 for the indicated time periods. TUNEL and confocal microscopy were used to detect apoptosis. Caspase-3 activation was examined by western blot analysis. Expression levels of DNA fragmation factor 40 （DFF40） were detected by RT-PCR. Results ONO-AE-248-induced neutro- phil death was negative in TUNEL assay. Western blot showed that ONO-AE-248 did not affect pro-caspase- 3 degradation. RT-PCR showed that ONO-AE-248 caused the down-regulation of DFF40 expression. Conclusion The experimental data suggested that the way of DNA fragmentation in the ONO-AE-248-induced neutrophil death is different from apoptosis. The down-regulation of DFF40 expression is likely to be one of explanations for the lack of the oligonucleosomal DNA fragmentation in the neutrophil death.