抗霉素A直接刺激线粒体所引起的超氧阴离子含量和膜电位变化的单线粒体水平研究

Abstract

建立了抗霉素A直接刺激提纯线粒体所引起的超氧阴离子(O2-.)含量和膜电位变化的单线粒体水平快速测定方法.利用杜恩斯(dOunCE)匀浆装置,结合差速离心法,从HElA细胞中提纯线粒体,经抗霉素A刺激和特异性荧光探针染色后,采用实验室自行研制的超高灵敏流式检测装置对线粒体进行快速、灵敏地逐一检测.与此同时,利用传统流式细胞仪在细胞水平对抗霉素A刺激所引起的O2-.含量和线粒体膜电位的变化进行了测定.实验结果表明,抗霉素A直接刺激线粒体能引起O2-.含量增加36%,膜电位下降16%;而抗霉素A在细胞水平能使得细胞中O2-.含量增加约3倍,膜电位的荧光信号增加1倍.研究结果表明,抗霉素A能直接诱导线粒体产生O2-.并引起膜电位的小幅度下降,所建立的线粒体活性参数微弱信号检测的新方法将有助于更深入地了解线粒体的功能以及药物的作用机理.

This research has established a sensitive method to measure superoxide anion production and mitochondrial membrane potential(MMP) of isolated mitochondria upon antimycin A stimulation.By combining Dounce homogenization and differential centrifugation,mitochondria were isolated from HeLa cells and treated with antimycin A.Then specific fluorescent probes were used to label mitochondria,and a laboratory-built high sensitivity flow cytometer(HSFCM) was used to detect superoxide anion and MMP at the single-mitochondrion level.For a comparison,conventional flow cytometry(FCM) was utilized to measure superoxide anion level and MMP of HeLa cells upon antimycin A treatment at the single-cell level.The results showed that upon drug stimulation,the superoxide anion level in the isolated mitochondria was increased by 36% and the MMP was decreased by 16%.The superoxide anion level in HeLa cells was increased about three times and the MMP was doubled upon antimycin A treatment.This experiment demonstrated that we have built a sensitive method that can sensitively detect the production of superoxide anions and the change of MMP at the single-mitochondrion level.