Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death
Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death
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DOI：
10.1023/a:1009604510329
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发表时间：
1999-12-01
期刊：
影响因子：
7.2
通讯作者：
Vermes, I
中科院分区：
文献类型：
--
作者：
Overbeeke, R;Yildirim, M;Vermes, I
在Jurkat和HSB 2-细胞中，通过流式细胞术监测诱导细胞死亡后连续发生的质膜和线粒体膜改变、细胞内pH变化和细胞内Ca 2+浓度变化。通过用抗Fas抗体处理或通过照射诱导细胞死亡。磷脂酰丝氨酸（PS）暴露和质膜完整性与FITC-膜联蛋白V粘附和碘化丙啶排斥。通过DiOC（6）荧光衰减的发生监测线粒体膜电位的转变。通过SNARF-1-AM在575 nm和635 nm处的荧光比率的变化来监测细胞内pH变化。Fura红猝灭法测定细胞内Ca ~（2+）浓度，发现Jurkat细胞对Fas抗体敏感，而HSB-2细胞对Fas抗体不敏感。HSB-2细胞比Jurkat细胞对辐射损伤更敏感，在所有实验中，线粒体膜电位的转变首先发生，几乎紧随PS暴露之后。细胞内Ca 2+浓度的波动发生较晚，不那么直言不讳。细胞内pH值的下降发生不早于抗Fas治疗后24小时。BAPTA-AM螯合细胞内Ca ~（2+）浓度对细胞死亡相关事件的时间顺序没有影响。
The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca2+ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin V adhesion and by Propidium Iodide exclusion. Transition of the mitochondrial membrane potential was monitored by the occurrence of decay of DiOC(6) fluorescence. Intracellular pH shifts were monitored by changes in the ratio of fluorescence at 575 nm and at 635 nm of SNARF-1-AM. Fluctuations in intracellular Ca2+ concentration were established by changes in Fura red quenching.The Jurkat cells were sensitive to anti-Fas treatment, while HSB-2 cells were not. HSB-2 cells appeared more sensitive to radiation damage than Jurkat cells.In all experiments the transition of mitochondrial membrane potential occurred first, almost immediately followed by PS exposure. Fluctuations in intracellular Ca2+ concentration occurred later and were less outspoken. A decrease in intracellular pH occurred not earlier than 24 hours after anti-Fas treatment. Chelation of intracellular Ca2+ concentration with BAPTA-AM had no effect on the time sequence of cell death related events.