Mito-OS-Timer:一种靶向监测线粒体氧化应激的荧光秒表

Mito-OS-Timer:A Targeted Fluorescent Stopwatch for Monitoring Mitochondrial Oxidative Stress

线粒体氧化应激(mitochondrial oxidative stress,Mito-OS)是一种线粒体内活性氧产生与抗氧化系统失衡状态,活性氧簇(reactive oxygen species,ROS)诱发氧化应激会造成线粒体损伤,被认为是促发衰老和疾病的一个重要因素。目前,氧化应激细胞或动物模型的评价方法主要基于细胞形态、动物表型或特征代谢产物产生情况等指标,无法实时监测动态变化。本研究建立了一种靶向线粒体的氧化应激荧光蛋白监测系统,命名为Mito-OS-Timer,可实时监测线粒体氧化应激动态变化。主要基于荧光蛋白DsRed1-E5红绿荧光转变速率与氧浓度变化呈正相关机理,将DsRed1-E5基因与定位线粒体内膜的ATP合酶亚基(ATP5PB片段)进行基因融合,构建了pMito-OS-Timer重组质粒以及HEK293T稳定表达细胞株,0-300μmol/L H_(2)O_(2)和0-5μmol/L鱼藤酮分别诱导处理后,结果显示细胞模型线粒体内红绿荧光转变速率与线粒体氧化应激程度呈现明显正相关。另外,利用Mito-OS-Timer检测pLVX-shFLCN沉默folliculin(FLCN)基因诱导氧化应激程度增强。此系统为研究线粒体氧化应激提供了一种新的可视化方法。

Mitochondrial oxidative stress(Mito-OS)is a kind of imbalance between reactive oxygen species production and antioxidant system in mitochondria.And oxidative stress induced by reactive oxygen species(ROS)is considered as important factors for aging and disease.At present,the evaluation methods of oxidative stress in cells or animal models are mainly based on cell morphology,animal phenotype or production of characteristic metabolites,et al.It is unable to monitor dynamic changes in real time.This study established a mitochondrial oxidative stress fluorescent protein monitoring system,named Mito-OS-Timer,which can monitor the dynamic changes of mitochondrial oxidative stress in real time.Based on the mechanism that dsRed1-E5 red/green fluorescence conversion rate was positively correlated with oxygen concentration change,dsRed1-E5 gene was fused with ATP5PB fragment of ATP synthase subunit located in mitochondrial inner membrane.Then,we established a recombinant plasmid pMito-OS-Timer and stably expressed cell line HEK293T.After being treated with 0-300μmol/L H_(2)O_(2) and 0-5μmol/L rotenone,the results showed that there was a significantly positive correlation between the rate of red-green fluorescence transformation and the degree of mitochondrial oxidative stress.In addition,Mito-OS-Timer was used to detect the enhancement of oxidative stress induced by folliculin(FLCN)gene silencing by pLVX-shFLCN.This system provides a new visualization method for studying mitochondrial oxidative stress.