基于超氧阴离子探针观察原位软骨细胞线粒体及线粒体炫  

Real-time Monitoring of Mitochondria and Mitochondrial Flashes of Chondrocytes in Situ Based on Superoxide Anion Probe

作　　者：胡晓青[1] 程锦[1] 张辛[1] 敖英芳[1] HU Xiao-Qing;CHENG Jin;ZHANG Xin;AO Ying-Fang(Beijing Key Laboratory of Sports Injuries,Peking University Third Hospital,Beijing 100191,China)

机构地区：[1]北京大学第三医院运动医学关节伤病北京市重点实验室,北京100191

出　　处：《中国生物化学与分子生物学报》2020年第7期858-864,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.81672212)资助。

摘　　要：关节软骨是无血供组织。软骨细胞处于低氧环境中,细胞能量来源的大部分来自糖酵解,仅有一小部分来自线粒体氧化磷酸化。因此,软骨细胞中线粒体的功能一直未被重视,且局限于体外细胞研究。本文采用激光共聚焦显微成像技术建立了原位软骨细胞及其线粒体形态的观察方法,发现软骨细胞形态及线粒体密度与细胞所在层次相关。有文献表明,软骨细胞内线粒体源性活性氧(reactive oxygen species,ROS)可与促炎因子共同作用加速软骨组织退变。线粒体呼吸链电子漏产生的超氧阴离子是细胞内ROS的最主要来源。而超氧阴离子半衰期非常短,并且很容易自发地转化为过氧化氢,使它成为最难检测的ROS信号。新型超氧阴离子探针mt-cpYFP(mitochondrially targeted circularly permuted yellow fluorescent protein)是一种可特异性感受超氧阴离子的黄色荧光蛋白。本文基于mt-cpYFP转基因小鼠实时监测软骨细胞内超氧阴离子变化情况,发现原位软骨细胞在静息状态下存在量子化超氧爆发现象(线粒体炫);同时,促炎因子白细胞介素-1β(interleukin-1β,IL-1β)刺激软骨细胞后,线粒体炫活性增加。因此,我们建立了原位软骨细胞线粒体形态与超氧阴离子动态变化的检测方法,并进一步探讨了促炎因子对关节软骨细胞线粒体ROS产生的影响。Although chondrocyte energy metabolism is dominantly glycolytic rather than through oxidative phosphorylation,mitochondrial dysfunction has been reported to be involved in the process of cartilage degeneration.Mitochondria in articular chondrocytes did not get much attention because of avascular and physiologically hypoxic environment in cartilage.Previous studies were limited to observe the mitochondrial morphology and superoxide anion of primary chondrocytes isolated and cultured in vitro,which were significantly different from in-situ chondrocytes in terms of morphological and metabolic characteristics.We established a set of novel in situ optical imaging approaches,and found that mitochondrial abundance as well as chondrocyte density declines with distance from the surface of intact cartilage.Other study has shown that in chondrocytes,mitochondrial derived reactive oxygen species(ROS)can work with pro-inflammatory cytokines to accelerate cartilage degeneration.Superoxide anion produced by electron leakage in mitochondrial respiratory chain is the most important source of intracellular ROS,and superoxide anion has a very short half-life and is easily converted to hydrogen peroxide spontaneously,making it the most difficult ROS signal to detect.This study was undertaken to make a morphometric analysis of mitochondria in articular chondrocytes using confocal micrographs and fluorescent probes,and to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.The results suggested that mitochondria were probably one of the essential organelles for articular chondrocytes.The superoxide flash response to proinflammatory cytokine stimulation suggests that chondrocyte mitochondria are a significant source of cellular oxidant and an important mediator which was previously under-appreciated in inflammatory cartilage diseases.

关 键 词：原位软骨细胞 线粒体炫 超氧阴离子 

分 类 号：R34[医药卫生—基础医学] R683