过氧亚硝基阴离子诱导大鼠气道上皮细胞凋亡及其机制探讨

APOPTOSIS OF RAT TRACHEAL EPITHELIAL CELLS INDUCED BY PEROXYNITRITE AND ITS MECHANISM

目的观察过氧亚硝基阴离子(peroxymitrite,ONOO^-)对大鼠气道上皮(rat tracheal epithelial,RTE)细胞凋亡的影响并探讨其作用机制。方法体外培养RTE细胞,外源性给予不同浓度ONOO^-(0.25、0.50、1.00mmol/L),培养1、6、12 h后,用流式细胞仪检测ONOO^-对RTE细胞凋亡百分率的影响、透射电镜观察细胞形态学变化、琼脂糖凝胶电泳观察DNA裂解情况。结果不同浓度ONOO均以时间依赖方式引起RTE细胞凋亡;透射电镜观察到染色质浓缩并凝集成块,核固缩以及凋亡小体形成等;琼脂糖凝胶电泳可见典型的DNA梯状条带出现。半胱天冬氨酸蛋白酶3(caspase-3)抑制剂Ac-DEVD-CHO可明显抑制ONOO^-诱导的RTE细胞凋亡。结论 ONOO^-以时间依赖方式引起培养的RTE细胞发生凋亡。caspase-3活化在ONOO^-诱导RTE细胞凋亡中起着重要作用。

Objective To investigate the effect of peroxynitrite on rats tracheal epithelial cells apoptosis and its mechanism.Methods After exposure of rats tracheal epithelial cells to 0.25、0.50 and 1.00 mmol/L peroxynitrite(ONOO^-)for 1,6 and 12 h,the apoptotic percentage was measured by flow cytometry;morphological alterations were observed by transmission electron microscope;specific DNA ladder pattern was detected by agarose gel electrophoresis. Results The apoptotic percentage was significantly increased when RTE cells exposed to various concentrations of ONOO^-(0.25、0.50 and 1.00 mmol/L)compared with vehicle group at all time points tested(1,6 and 12 h)(P<0.01,respectively).ONOO^-caused an increase of apoptotic percentage in RTE cells in a time-dependent manner at different concentrations.After exposure of RTE cells to ONOO^-(0.5 mmol/L)for 6 h and 12 h,RTE cells showed specific DNA ladder pattern in agarose gel electrophoresis.Characteristic morphological alterations including cell shrinkage,chromatin condensation were observed by transmission electron microscope.Caspase-3 inhibitor Ac-DEVD-CHO obviously prevented the apoptosis of RTE cells induced by ONOO-. Specific DNA ladder pattern induced by ONOO^-were not observed in agarose gel electrophoresis after treatment with Ac-DEVD-CHO(10 μmol/L).Conclusion These results suggested that ONOO^-could cause apoptosis in cultured RTE cells in a time-dependent manner.The activation of caspase-3 played a crucial role in the apoptosis of RTE cells induced by ONOO^-.