Quantitative Measurement of Mitochondrial Fusion in vitro by High-Sensitivity Flow Cytometry

线粒体是高度动态的细胞器，在真核细胞能量代谢和信号转导中发挥重要的调控作用。线粒体融合分裂与细胞代谢、线粒体自噬、细胞凋亡等多种生命活动息息相关，融合分裂失衡会导致多种人类疾病，包括神经退行性疾病、心血管疾病、癌症等。因此，建立快速的线粒体融合检测方法并对该过程进行定量检测显得尤为重要。目前线粒体融合的检测手段主要是采用荧光显微镜和透射电子显微镜对线粒体融合现象进行观察，但这两种方法都在检测速度和定量方面存在很大的局限性。线粒体融合的相关研究迫切需要发展高灵敏、高通量的单线粒体水平检测技术。 结合瑞利散射和鞘流单分子荧光检测技术，我们课题组研发了超高灵敏流式检测技术（highsensitiv...Mitochondria play a central role in the regulation of energy metabolism and signal transduction in eukaryotic cells. Besides, mitochondria are highly dynamic organelles that continually undergo fusion and fission. Mitochondrial dynamics actively contributes to a lot of cellular activities, such as cell metabolism, mitophagy and apoptosis, and dysfunction of which will lead to several human disease...学位：理学硕士院系专业：化学化工学院_化学生物学学号：2052013115166

Recent Advances in Mitochondrial Proteomics

线粒体是细胞能量代谢、生物合成和细胞死亡的调控中心,其功能异常会导致许多疾病的产生。线粒体蛋白质组学研究为解析线粒体的生理功能、探索线粒体相关疾病的发病机制及促进线粒体为靶向的药物研发提供重要理论依据。本文对线粒体蛋白质组学的研究方法,尤其是近年的技术发展以及线粒体蛋白质组学的性质及其应用进行总结,并对其未来的发展前景和挑战进行展望。Mitochondria play a central role in the regulation of cellular energy metabolism,bio-synthesis and cell death.Dysfunction of mitochondria can lead to many diseases.Mitochondrial proteomics provides important theoretical foundation for a systematic understanding of the biological functions of mitochondria,studying the mechanisms of mitochondria-related diseases,and promoting the research and development of mitochondria-targeting drugs.The methodologies,recent technology development,and characteristics and applications of mitochondrial proteomics were reviewed and the challenges and prospects were also discussed.国家自然科学基金(Nos.91313302;90913015;21225523)资助项目~

High-Throughput Assessment of Mitochondrial Fluorescence Labeling at Single-Particle Level

线粒体是真核细胞能量代谢和信号转导的调控中枢,虽然各种灵敏、特异的线粒体荧光标记技术已经被广泛应用于线粒体研究,但仍缺乏单线粒体水平的荧光染色性能评估方法。基于超高灵敏流式检测技术(High sensitivity flow cytometry,HSFCM)能对单个线粒体进行高灵敏、高通量、多参数定量分析的独特优势,本研究发展了一种单线粒体水平的荧光标记高通量评估方法。将携带靶向线粒体绿色荧光蛋白基因的p Ac GFP1-Mito质粒转染至人宫颈癌He La细胞中,用G418筛选出稳定转染细胞,分别从瞬时转染和稳定转染的细胞中提取线粒体。此外,从未转染质粒的正常He La细胞中提取线粒体,分别进行Mito Tracker Green标记以及SYTO 62线粒体DNA染色,应用实验室自行研制的超高灵敏流式检测装置在单线粒体水平对这4种线粒体标记方法的荧光亮度、标记效率和稳定性进行评估。实验结果表明,稳定转染细胞中单个线粒体的绿色荧光蛋白(GFP)荧光亮度为瞬时转染的17.7倍,比Mito Tracker Green标记的线粒体亮度高约两个数量级,且标记稳定性好。本研究为线粒体标记方法的选择提供了一种先进的分析方法。Mitochondria play a central role in the regulation of energy metabolism and signal transduction in eukaryotic cells. Although many fluorescent labeling strategies have been developed for mitochondrial studies,the methods that enable labeling efficiency assessment at the single-mitochondrion level are still lacking. By employing the unique advantages of high sensitivity flow cytometry( HSFCM) in the sensitive,rapid,and quantitative multiparameter analysis of individual mitochondria,here we examined the performance of several different mitochondrial labeling strategies from the perspectives of brightness,labeling ratio,and stability.Mitochondria isolated from He La cells transfected with p Ac GFP1-Mito plasmid upon transient or stable transfections,and mitochondria directly labeled with Mito Tracker Green or SYTO 62 were analyzed by a laboratory-built three-channel HSFCM. Upon the quantitative measurement of fluorescence brightness,it was found that the fluorescence intensity of green fluorescent protein( GFP) in mitochondria isolated from cells with stable transfection was about 17. 7-fold higher than the transient transfection ones,and was approximately two orders of magnitude brighter than mitochondria labeled with Mito Tracker Green. On the other hand,the fluorescence signal of SYTO 62 labeling decreased upon washing,indicating its rapid dissociation rate. The strong fluorescence intensity and good labeling stability make stable transfection an efficient method to label mitochondria. The experimental results demonstrates that HSFCM provides a powerful analytical tool to assess the performance of mitochondrial fluorescence labeling via high throughput single mitochondria analysis.国家自然科学基金项目(Nos.91313302,90913015,21225523)资助~

驼绒藜属牧草种子生物学特性、种子生产技术及基地建设

该项研究针对具有广泛开发利用前景的驼绒藜属牧草种子存在的寿命短，易于劣变等问题进行了种子生物学特性的系统研究，包括种子的发育、萌发、贮藏、活力、营养代谢和激素调节、抗逆性以及遗传差异等；探索了驼绒藜属植物“短命种子”的发育规律和劣变机理，提出的种子超干燥贮藏技术具有创新性；首次系统研究并提出的驼绒藜属种子生产和贮藏技术体系，可提高种子产量3～5倍，延长种子寿命1～2年，秋季种苗移栽定植技术成活率达80%以上，比传统技术提高50%以上；建立了华北驼绒藜人工种子生产基地1000亩、天然种群原种基地2000亩，年产种子15吨，对恢复和保护利用驼绒藜属植物资源具有重要意义