单细胞组学技术及其在植物保卫细胞研究中的应用

单细胞组学技术在动物研究中已经得到广泛应用,但在植物学领域尤其是保卫细胞研究中还处于起步阶段。由保卫细胞构成的气孔承担着植物生命过程中水分散发及气体交换大门的作用。将单细胞组学技术应用到保卫细胞功能解析中将有助于了解保卫细胞参与的基本生理过程。该文综述了植物单细胞组学技术的发展、保卫细胞研究现状及单细胞组学技术在植物保卫细胞研究中的初步应用,为借助该技术解决植物生物学中保卫细胞发育、代谢及对环境胁迫响应等基本问题提供研究思路和方法。     

空间转录物组学技术进展及其在神经科学领域中的应用

空间转录物组学是在单细胞RNA测序技术基础上实现细胞空间位置信息测定的组学技术。该技术克服了单细胞转录物组学在单细胞分离建库过程中丢失细胞在组织中空间信息的问题,可同时提供研究对象的转录物组数据信息和在组织中的空间位置信息。空间转录物组学技术对研究细胞谱系的发生过程、细胞间的调控机制和相互作用等具有重要作用,是组学技术研究的重要发展方向和热点。近年来,空间转录物组学技术发展迅速,新的检测方法不断产生,检测灵敏度、分辨率和检测通量等技术指标不断提升。本文根据获取空间信息的原理不同,将较为常用的空间转录物组学技术进行了分类,总结了各类方法的检测原理、代表性技术手段及其相应的技术指标。随后,从脑细胞类型区分与细胞层图谱构建、神经系统相关疾病特征分析与标志物研究两个方面举例论述了空间转录物组学技术在神经科学中的应用。最后,对空间转录物组学技术目前存在的问题进行了总结,并对其未来的发展方向进行了展望。     

单细胞测序技术在干细胞领域的研究进展

细胞异质性是生物体内普遍存在的一种特性,这种特性容易受外界因素的影响,甚至是单一类型的细胞在生长环境发生改变时,其基因表达也可能出现变化并产生差异。干细胞是一类具有无限自我更新和分化潜能的特殊类型的细胞,在胚胎组织发育和成体组织的动态平衡中发挥了重要作用。单细胞测序为分析包括干细胞在内的细胞异质性提供了强有力的工具,这种技术可通过更加准确的方式剖析细胞异质性。该文综述了近年来发展起来的单细胞测序技术,包括单细胞分离、基因组扩增和测序分析,并讨论了它们在干细胞(包括多能干细胞、肿瘤干细胞和组织特异性干细胞)研究中的应用。     

单细胞转录组测序技术在动物上的应用研究

细胞是机体最基本的结构组成及功能单位,细胞类型和功能由其整个转录表达谱决定,通过单细胞转录组测序可以获得单个细胞转录表达谱,由此以高精度分辨率鉴定细胞类型、细胞状态以及稀有类型细胞,从而可以在单细胞水平分析细胞动态变化及细胞间的关系,深入解析驱动细胞变化及细胞异常背后的分子细胞机制.随着单细胞测序技术稳定性和测序通量的...     

单细胞多组学技术新进展及其在发育生物学研究中的应用

单细胞多组学技术基于单细胞测序技术和高通量组学方法发展而来,能在单细胞分辨率下同时整合转录组、基因组、表观遗传组、蛋白质组或空间状态等两种或多种组学信息.相比单细胞单组学技术,单细胞多组学技术能多层次、多角度地描绘细胞间的异质性,挖掘各层组学之间的直接和潜在联系,从而更加全面和系统地描绘细胞的状态和命运,在生命科学和医学多领域有广泛的应用前景.本文总结了现有的单细胞多组学技术,预测了其发展趋势,讨论了其在发育生物学中的应用实例和潜力.     

单细胞转录组技术及其在人类细胞图谱构建中的应用

单细胞转录组技术在单细胞水平上进行转录组测序,提供了单个细胞的基因表达差异信息,使在单细胞尺度下研究个体细胞、相关环境细胞及其相互作用的机理成为可能.近年来,单细胞转录组技术在c DNA扩增原理上经历了从末端加尾、体外逆转录到模板置换的方法发展,大大提高了基因检测的数量、基因表达的准确性等.同时,在单细胞选取方式上进行了从96/384孔板到油包水液滴以及纳米微孔的创新,在提高通量和重复性的同时降低了整体实验成本.单细胞转录组技术广泛应用于细胞群体分类和异质性研究,推动了从发育生物学到正常、病态组织细胞图谱的构建.本文对单细胞转录组技术近年的技术进展以及在人类细胞图谱构建中的应用进行了综述.     

激光捕获显微切割在肿瘤多组学研究中的应用进展

当今社会,肿瘤因其高发病率和高死亡率成为威胁人类健康的重要疾病,研究者们对其发病机制及治疗手段的研究和探索也在不断深入。随着单细胞多组学测序技术的发展,肿瘤组织的异质性问题逐渐被研究人员所认识。为了解决这一问题,激光捕获显微切割（laser capture microdissection,LCM）技术应运而生。LCM技术是一种在显微镜直视下从器官或组织中准确获取某种特定的细胞群或单个细胞的样本收集技术。LCM技术结合多种分子生物学手段可以对异质性组织进行多组学研究,丰富了现有的肿瘤蛋白质组学、基因组学以及转录组学图谱,因此,LCM技术成为研究特异性表达及分子机制的有力工具,在肿瘤学领域得到广泛应用。基于此,对LCM的原理、优势及其在肿瘤多组学研究中的应用进行了综述,并对其未来可能的发展方向进行了展望,以期为肿瘤的研究和治疗提供新的思路。     

单细胞转录组测序技术在细胞分类中的应用

多细胞有机体的细胞类型多且复杂,细胞间普遍存在异质性。目前,单细胞转录组测序(single-cell RNA sequencing,scRNA-seq)技术是一项新兴的研究单个细胞转录水平的技术,其从数千个平行的细胞中生成转录谱,揭示个体细胞基因组的差异性表达,反映细胞间的异质性,从而鉴定出不同细胞类型,形成组织或器官的细胞图谱,在生物和临床医学等领域发挥重要作用。该文在对scRNA-seq测序平台进行阐述和比较的基础上,着重介绍其在神经系统和免疫系统细胞类型探索中的应用,并且总结scRNA-seq与空间转录组技术相结合的研究成果。     

紫菜叶状体细胞的酶法分离及其养殖研究

本文应用生物技术方法,进行紫菜细胞的菜苗和海上养殖已获得成功。用海螺酶将紫菜叶状体细胞分离成单细胞和原生质体,研究了叶状体的不同细胞类型的再生和发育。不同海水比重、不同温度对细胞的分离和培养的影响,并研究了单细胞和原生质体的附着及其海上的养殖。紫菜酶法采苗的成功,将会对传统的紫菜养殖产生根本的变革。     

单细胞拉曼光谱在微生物研究中的应用

拉曼显微光谱是一种能够提供0.5–1.0μm空间分辨率的单个微生物细胞内化学结构信息的研究技术。近几年来,拉曼显微光谱被越来越多地应用于微生物单细胞的研究中,它可以快速无损地检测微生物细胞内的特征化学组分。典型的单个微生物细胞的拉曼光谱包含核酸、蛋白质、碳水化合物、脂质和色素(例如类胡萝卜素)等信息,这些信息能够表征微生物细胞的基因型、表型和生理状态。所以单细胞拉曼显微光谱是一种可用于区分微生物样品的"全生物指纹"技术,它可用于研究单个微生物细胞生命阶段的转变、鉴定微生物单细胞中的色素及其他化合物的含量变化等。本文综述了目前拉曼显微光谱在微生物单细胞研究上的应用,主要包括与稳定同位素标记(stable isotope probing,SIP)、拉曼成像、光谱分类和细胞分选技术结合来探究微生物单细胞对物质吸收后特征峰的变化、推导物质循环过程、进行微生物分类鉴定和探索基因型与表型的关系。拉曼显微光谱作为微生物单细胞研究的手段之一,在代谢过程的研究、活细胞分选和细胞对物质的利用上具有广泛的应用前景。     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

微生物单细胞基因组技术及其在环境微生物研究中的应用

单细胞及单细胞基因组学研究是近年生命科学研究的热点之一,微生物单细胞基因组学研究是继微生物元基因组学(又称宏基因组学,Metagenomics)之后新发展起来的,可有效获取环境中大量无法培养的微生物遗传信息的技术。微生物单细胞基因组技术包括单细胞获取、全基因组扩增、全基因组测序以及数据分析等步骤,目前该技术在环境微生物研究中的应用主要集中于探索未被元基因组技术或其它常规技术探测到的新型功能基因,或是对环境中物种丰度极小的未培养微生物的发现,以及对微生物细胞生命进化过程的研究等。本文对微生物单细胞基因组技术中单细胞获取和全基因组扩增所涉及到的不同方法以及应用此技术对环境微生物取得的主要研究进展进行综述。     

Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices

Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. However, since variability exists between individual cells even in the same kind of cells, increased emphasis has been put on the analysis of individual cells for getting better understanding on the organism functions. During the past two decades, various techniques have been developed for single-cell analysis. Capillary electrophoresis is an excellent technique for identifying and quantifying the contents of single cells. The microfluidic devices afford a versatile platform for single-cell analysis owing to their unique characteristics. This article provides a review on recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices; focus areas to be covered include sampling techniques, detection methods and main applications in capillary electrophoresis, and cell culture, cell manipulation, chemical cytometry and cellular physiology on microfluidic devices.     

Single-cell analysis and isolation for microbiology and biotechnology: methods and applications

Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.     

Metabolite analyses of single cells

Single-cell analysis is a promising method for understanding not only cellular physiology but also biological mechanisms of multicellular organisms. Although neighboring cells in multicellular organisms originate from the same genomic information, different circumstances around cells or epigenetic differences have different influences on each cell, leading to differing expression of genes, and thus differing levels and dynamics of metabolites, in single cells. However, single-cell analysis is a tough challenge, even with recent technologies, because of the small size of single cells. Unlike genes, metabolites cannot be amplified, and therefore metabolite analysis is another issue. To analyze such a tiny quantity of metabolites in a single cell, various techniques have been tried and developed. Especially in mass spectrometry, marked improvements in both detection sensitivity and ionization techniques have opened up the challenge for the analysis of metabolites in single cells. In this review, we discuss the method for metabolite detection at the level of single cells and recent advancements in technology.     

The stochastic nature of biochemical networks

Cell behaviour and the cellular environment are stochastic. Phenotypes vary across isogenic populations and in individual cells over time. Here we will argue that to understand the abilities of cells we need to understand their stochastic nature. New experimental techniques allow gene expression to be followed in single cells over time and reveal stochastic bursts of both mRNA and protein synthesis in many different types of organisms. Stochasticity has been shown to be exploited by bacteria and viruses to decide between different behaviours. In fluctuating environments, cells that respond stochastically can out-compete those that sense environmental changes, and stochasticity may even have contributed to chromosomal gene order. We will focus on advances in modelling stochasticity, in understanding its effects on evolution and cellular design, and on means by which it may be exploited in biotechnology and medicine.