Multiparameter Quantification of Lipid Nanomedicines by High Sensitivity Flow Cytometry

纳米药物因其在靶向给药、药物缓控释、提高难溶性药物与多肽药物的生物 利用度、降低药物毒副作用等方面显示出的良好应用前景，而成为21世纪生物 医学领域的重要发展方向。纳米药物的载体包括高分子纳米颗粒、脂质纳米颗粒、 无机纳米颗粒、病毒类纳米颗粒等，其中以磷脂为主要骨架基元的脂质纳米药物 （主要包括脂质体、实心脂质纳米粒、磷脂胶束和细胞分泌囊泡等）具有良好的 稳定性、优异的载药量和生物相容性，是目前临床上最受认可的载药系统。 为了加快脂质纳米药物的研发进程并保障临床应用的药效，必须对纳米颗粒 的粒径及其分布、颗粒浓度、药物包封率、载药量、表面配体数量及其分布等物 理、化学性状进行快...The growing interest in the biomedical application of nanomedicine is largely attributable to its unique and appealing features in targeted delivery, controlled release and improved bioavailability. Currently, nanomedicines can be broadly divided into the following categories: lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles and virus nanoparticles. Among them, lipid based...学位：理学博士院系专业：化学化工学院_化学生物学学号：2052014015396

Study the Effect of Antimycin A on Superoxide Anion Production and Mitochondrial Membrane Potential of Isolated Mitochondria at the Single-mitochondrion Level

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.国家自然科学基金项目(90913015); 教育部博士点基金(20090121110009

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~

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~

一种磁性纳米粒子及其制备方法和应用

本发明涉及一种磁性纳米粒子及其制备方法和应用。具体地，所述磁性纳米粒子具有下组特征：1)所述磁性纳米粒子为铁氧体；2)所述磁性纳米粒子的粒径为0.1-30nm；3)所述磁性纳米粒子的弛豫率r1≥1.2mM-1s-1；4)所述磁性纳米粒子的驰豫率比r2-r1≤3.5且r2-r1≥1.05；5)0.5mM所述磁性纳米粒子的T1加权信号强度≥70。本发明还公开了所述磁性纳米粒子的制备方法和应用。将所述磁性纳米粒子应用于磁共振成像中可获得一种成像性能显著优异、磁敏感性高且T1信号丰富的优质MRI造影剂，可提高其对肿瘤、心脑血管等重大疾病的发现与检出，从而显著降低医学检测和治疗成本

纤维素/陶瓷复合膜的制备及油水分离性能研究

目的提高陶瓷膜对水包油乳化液的分离能力。方法将纤维素粉末充分溶解在氢氧化钠、尿素、水配制的溶解液中,低温真空下在多孔陶瓷分离膜表面构筑亲水性纤维素涂层。通过X射线衍射仪(XRD)、场发射扫描电子显微镜(SEM)、接触角测量仪、紫外-可见分光光度计表征纤维素/陶瓷复合膜的组成、结构、表面润湿性及水包油(甲苯)乳液分离效率。结果纤维素成功复合到多孔陶瓷膜孔隙表面,空气中水滴在膜表面的接触角为0°,水中油滴的接触角高达155°左右,使多孔陶瓷膜具备了在空气中超亲水-水下超疏油的特性。通过油水分离实验发现,在常温常压下,多孔陶瓷膜对水包甲苯乳液高效分离,其油水分离能力(>99.26%)、抗污染性(经20次循环分离后,三种复合的平板陶瓷膜的水通量变化不大)得到显著提高,表现出较好的油水分离性能。结论陶瓷分离膜经纤维素修饰后,表面亲水性和水下超疏油性都得到了提高,油水分离能力可达99.26%,重复使用20次的纯水通量无明显衰减

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel