MitoTracker对在体小鼠小脑和体外培养颗粒细胞线粒体的染色方法

目的：采用线粒体红色荧光探针MitoTracker标记小脑颗粒细胞线粒体，并与标记体外培养神经元线粒体进行比较，探讨MitoTracker标记在体脑细胞线粒体的实验方法。方法：在脑立体定位仪下，微量注射MitoTracker线粒体红色荧光探针到小鼠小脑皮质，继续饲养3d后，常规取脑、固定、切片，利用共聚焦显微镜观察线粒体标记情况。结果：共聚焦显微镜下，可观察到在体标记的神经细胞内颗粒样线粒体呈团块状分布，排列较体外培养神经元标记的杆状线粒体紧密。结论：MitoTracker线粒体红色荧光探针可用于标记在体小脑细胞的线粒体。

In vivo 3-photon fluorescence microscopy of white matter in mouse brain excited at the 1700 nm window

White matter,a densely packed collection of myelinated axons,plays an essential part in neural networks.With high spatial resolution and deep penetration,multi-photon microscopy(MPM)is promising for white matter imaging in animal models in vivo.The third harmonic generation(THG)signal can be generated from white matter,but the bottom part of the white matter layer generates weak THG due to its high scattering.Here,we demonstrate an in vivo labeling and imaging technology,capable of visualizing the white matter layer in the mouse brain,combining°uorescence labeling with MitoTracker Red and three-photon°uorescence(3PF)microscopy excited at the 1700 nm window.3PF signals are several times higher than THG signals,resulting in deeper imaging of the white matter layer with the former.Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.

A two-photon mitotracker based on a naphthalimide fluorophore:Synthesis, photophysical properties and cell imaging

PAHPN. a naphthalimide-based mitotracker with reasonable two-photon excitation emission activity and polarity-sensitive fluorescence properties has been efficiently synthesized and studied in twophoton, co-localization, and FLIM imaging.

地塞米松诱导小鼠胸腺细胞凋亡中线粒体质量的变化

目的研究地塞米松(DEX)诱导的小鼠胸腺细胞凋亡中线粒体质量变化。方法分别设对照组(Control)和DEX组;在6 h,分别以NAO和M itotracker G reen(MG)单染流式细胞术检测线粒体质量变化,以D iOC6(3)染色流式细胞术检测线粒体膜电势变化,以Annexin V-PE/MG双染流式细胞术检测细胞凋亡中线粒体质量改变特点。结果NAO染色检测结果显示终浓度1μmol.L-1DEX可以诱导小鼠胸腺细胞线粒体心磷脂含量降低,与对照组比较P<0.01;MG染色流式细胞术结果表明DEX(P<0.01)诱导了胸腺细胞线粒体质量的降低;DEX(P<0.01)诱导线粒体膜电势依赖性D iOC6(3)在胸腺细胞中可染性下降;Annexin V-PE/MG双染流式细胞术显示,凋亡细胞中存在一定比例的低线粒体质量细胞,DEX组该群细胞多于对照组(P<0.01)。结论DEX诱导小鼠胸腺细胞凋亡中线粒体质量降低;线粒体质量评价在细胞凋亡相关药理学研究领域有应用前景。

线粒体活性对人类精子活动力的影响

目的:分析线粒体活性对人类精子活动力的影响。方法:以线粒体特异性荧光染料--mitotracker deep red 633(MTDR对不同活力的精子进行染色,然后用流式细胞术测定精子线粒体活性(即MTDR荧光强度值)。用雷帕霉素(rapamycin,RP)和羟氯喹(hydroxychloroquineon,HCQ)调控精子活性水平,测定精子活动力的变化。结果:MTDR荧光强度值与前向运动速率(progressive motility,PR)不相关(r=0.182,P=0.268)。5 nmol/L和100 nmol/L的RP均可明显降低线粒体活性,50μmol/L HCQ可明显提高线粒体活性(P<0.05),但它们对精子活动力影响都不明显(P>0.05)。结论:线粒体活性对人类精子活力影响不明显。

Mitochondrial dysfunctions in neurodegenerative diseases:role in disease pathogenesis,strategies for analysis and therapeutic prospects

Fundamental organelles that occur in every cell type with the exception of mammal erythrocytes,the mitochondria are required for multiple pivotal processes that include the production of biological energy,the biosynthesis of reactive oxygen species,the control of calcium homeostasis,and the triggering of cell death.The disruption of anyone of these processes has been shown to impact strongly the function of all cells,but especially of neurons.In this review,we discuss the role of the mitochondria impairment in the development of the neurodegenerative diseases Amyotrophic Lateral Sclerosis,Parkinson's disease and Alzheimer's disease.We highlight how mitochondria disruption revolves around the processes that underlie the mitochondria's life cycle:fusion,fission,production of reactive oxygen species and energy failure.Both genetic and sporadic forms of neurodegenerative diseases are unavoidably accompanied with and often caused by the dysfunction in one or more of the key mitochondrial processes.Therefore,in order to get in depth insights into their health status in neurodegenerative diseases,we need to focus into innovative strategies aimed at characterizing the various mitochondrial processes.Current techniques include Mitostress,Mitotracker,transmission electron microscopy,oxidative stress assays along with expression measurement of the proteins that maintain the mitochondrial health.We will also discuss a panel of approaches aimed at mitigating the mitochondrial dysfunction.These include canonical drugs,natural compounds,supplements,lifestyle interventions and innovative approaches as mitochondria transplantation and gene therapy.In conclusion,because mitochondria are fundamental organelles necessary for virtually all the cell functions and are severely impaired in neurodegenerative diseases,it is critical to develop novel methods to measure the mitochondrial state,and novel therapeutic strategies aimed at improving their health.

细胞和组织中两种线粒体标记方法的比较

目的比较探针和抗线粒体蛋白抗体标记细胞及组织中线粒体的效果,为相关研究选取适合的标记方法提供参考。方法在经体积分数4%多聚甲醛固定的HepG2细胞中,分别使用100 nmol/L MitoTracker Deep Red探针和抗葡萄糖调节蛋白75(Grp75)抗体标记线粒体;在经体积分数4%多聚甲醛固定的HeLa细胞中,分别使用75 nmol/L、100 nmol/L MitoTracker Deep Red探针和抗Grp75抗体标记线粒体;取人肝组织冰冻切片,经4%多聚甲醛固定后,分别使用150 nmol/L MitoTracker Deep Red和抗Grp75抗体标记线粒体。共聚焦显微镜下观察。结果MitoTracker Deep Red探针标记细胞线粒体的效果不如抗Grp75抗体标记清晰,且在HeLa细胞中有非特异性染色,抗体标记可更清楚地反映线粒体的点状和管状分布;在组织中MitoTracker Deep Red探针标记的单个细胞更均匀,标记线粒体的特异性更高,效果优于抗体染色。结论在细胞中使用抗Grp75抗体标记线粒体效果更好,能直观反映线粒体的分裂融合状态;在组织中使用MitoTracker Deep Red染色效果更佳。