ONO-AE-248诱发中性粒细胞非凋亡性程序化细胞死亡的生化信号转导初探

目的：研究ONO-AE-248诱发中性粒细胞（PMN）非凋亡性程序化细胞死亡过程中，细胞死亡相关的蛋白分子caspase-3、caspase-8、p-38MAPK和PKC的作用，探讨这种细胞死亡的分子机制。方法：运用光镜和DNA电泳对PMN形态学变化和生化特征进行观察和评估；通过Western blot显示PMN中caspase-3、caspase-8活性改变和p-38MAPK磷酸化改变；运用MTS法检测PKC抑制剂对PMN活性的影响。结果：PMN经ONO-AE-248刺激，绝大多数分叶核融合成单叶核（12小时），DNA琼脂糖电泳结果显示缺乏梯状条带（24小时），而且ONO-AE-248对caspase-3、caspase-8活性和p-38MAPK磷酸化水平无明显影响。然而，PKC抑制剂STS和H-7能显著抑制ONO-AE-248对PMN的促死亡效应。结论：ONO-AE-248所诱导的PMN死亡可能是一种非凋亡性的主动性细胞死亡，即非凋亡性程序化细胞死亡。该死亡过程不依赖caspase-3、caspase-8的活化和p-38MAPK的磷酸化，但是PKC可能发挥正向的调控作用。

Studies on the transduction of biological signals of ONO-AE-248-induced non-apoptotic programmed cell death of neutrophils

Objective: To investigate the role of caspase-3, caspase-8, p-38MAPK and PKC in the death of neutrophils induced by ONO-AE-248. Methods: Light microsopy and agarose gel electrophoresis of low-molecular-weight DNA were used to observe the changes of morphology and DNA fragmentation. Western blot technique was used to detect the activity of caspase-3, caspase-8 and p-38MAPK. The effect of PKC inhibitors on the viability of neutrophils was determined by MTS assay. Results: ONO-AE-248 rapidly caused a unique form of neutrophil death. During these processes, morphological change of nucleus such as fusion of the lobules was apparent and DNA fragmentation was absent in ONO-AE-248-treated neutrophils. Western blot showed that ONO-AE-248 did not affect activation of caspase-3, caspase-8 and p-38MAPK. However, ONO-AE-248-induced neutrophil death was significantly aborted by two PKC inhibitors(staurosporine or H-7). Conclusion: ONO-AE-248 could induce a form of non-apoptotic programmed cell death of neutrophils, which might be mediated by PKC pathway and be independent on caspase-3, caspase-8 and p-38MAPK.