流式细胞术

流式细胞术发展简史 流式细胞术(flow cytometry,FCM)是一种可以对细胞或亚细胞结构进行快速测量的新型分析技术和分选技术.其特点是:①测量速度快,最快可在1s内计测数万个细胞;②可进行多参数测量,对同一个细胞做有关物理、化学特性的多参数测量,并具有明显的统计学意义;③是一门综合性的高科技方法,它综合了激光技术、计算机技术、流体力学、细胞化学、图像技术等众多领域的知识和成果;④既是细胞分析技术,又是精确的分选技术.     

流式细胞术在细菌快速检测中的应用

流式细胞仪(Flow cytometer)是集应用流体学、光学、电子学、生物学、免疫学等多门学科和技术于一体的新型高科技仪器。它的核心技术是流式细胞术(Flow cytometry,FCM),该技术是利用流式细胞仪,使单个细胞或其他微小生物粒子处于快速直线流动状态,且逐个通过光束,从而对单个细胞或微粒进行多参数(数量、大小、核酸含量、细胞活性、特定菌群或物种等)定量分析和分选的检测技术,具有快速、灵敏、精确以及便于操作等突出优点。本文简要介绍流式细胞仪的原理,并论述流式细胞技术在实验室研究、工业生产、临床诊断、环境评估等领域的细菌快速检测应用。     

流式细胞术的发展及在植物研究中的应用

流式细胞术是通过对液流中各种荧光标记的颗粒进行多参数快速高效的定性或定量测定的方法,在科学研究的多个领域发挥重要作用。然而,由于植物组织及细胞壁和次生代谢产物等细胞的特殊成分和结构,限制了其在植物研究领域的应用。本文在介绍流式细胞仪发展和组成分类的基础上,着重讨论了流式细胞术在植物领域的应用、研究进展及应用限制,进而展望该研究领域的发展趋势,为拓宽植物流式细胞术的潜在应用范围提供新的思考方向。     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒（如细胞,细菌）的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来：功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒(如细胞,细菌)的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来:功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

专家答读者问：流式细胞仪如何将不同类型的细胞分开

<正>流式细胞仪（flow cytometer）是一种在功能水平上对单细胞或其他生物粒子进行定量分析和分选的检测仪器，可以每秒钟分析上万个细胞/粒子，并能同时从一个细胞/粒子中获得多个参数。其应用范围非常广泛，而且还在不断拓展，细胞分选也是它的重要应用之一。它能够根据每个细胞的光散射和荧光特征，将特定的细胞从细胞群体中分选出来。下文以细胞为例具体说明流式细胞仪的工作原理、技术特点和应用。流式细胞仪能够利用细胞的光散射特征，     

质谱流式技术用于单细胞检测

质谱流式技术(mass cytometry)是利用质谱原理对单细胞进行多参数检测的流式技术,能够在单细胞水平实现超过50种标志物的同时测量,显著增强了对细胞生长进程和复杂细胞系统的评估能力。该文简要介绍了质谱流式技术的基本工作原理,并从金属元素标记、质量分析器、高维单细胞数据处理等方面展开论述,阐明设计新型金属元素标签和选择飞行时间质谱的必要性,归纳分析高维单细胞数据的算法并总结各种算法的优点和局限性。     

流式细胞术

流式细胞术是一种综合应用光学、机械学、流体力学、电子计算机、细胞生物学、分子免疫学等学科技术,对高速流动的细胞或亚细胞进行快速定量测定和分析的方法。它一秒钟能分析几千个细胞,并同时测定细胞的多个参数,广泛应用于生物医学的许多领域,如测定细胞的特征(形态、膜电位等)和细胞内pH,细胞DNA、蛋白质含量、表面受体、Ca2+等。对生物工程学来说,了解细胞的这些参数尤为重要,因为它们能比用传统技术测得的数据更好地描述细胞群体。从流式细胞仪对细胞多种参数的测定及原理,到它在生物工程学中的应用等方面进行了介绍,并讨论了流式细胞术的局限性和面临的挑战。     

流式细胞术揭示出枯草芽孢杆菌多态异质性

新近的研究发现,微生物群体异质性现象普遍存在,与微生物群体许多关键功能密切相关.微生物群体中的多种异质性状态需要单细胞水平的分析技术才能被揭示,流式细胞术是获取异质性状态精确分布的重要工具.但微生物细胞尺寸微小、生物分子含量少、常常缺乏特异性试剂等都限制着传统流式细胞技术在微生物研究领域的应用.本论文采用新型的低背景、高灵敏度和高分辨率流式细胞仪,以增强的前向散射光、侧向散射光以及紫外光激发的细菌自发荧光水平这三个无需任何荧光标记就可以检测的信号为参数,首次揭示出不同生长状态的枯草芽孢杆菌具有复杂、动态的异质性状态分布.这一方法鉴定出的枯草芽孢杆菌多种状态及其与生理功能相关的、高度关联的变化,可能对该菌的生理变化规律及其分子机理的认识提供新的机遇.本论文也讨论了这一采用新型高灵敏度、高分辨率流式细胞仪测量非标记细胞参数的方法对于广泛开展各种微生物多态性研究具有巨大潜力.     

流式细胞术在高等植物研究中的应用

流式细胞术(FCM)是根据所测定的各种细胞性质的不同组合,从细胞群体中把某个亚群分选出来,并对它的功能和形态学进行研究或进一步培养分析。流式细胞术具有快速、灵敏和同时进行多参数检测等优点,对其基本原理和在高等植物中的应用进行了介绍。     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

A chromium-tolerant plant growing in Cr-contaminated land

In this paper, a kind of Typhaceae plant species, Typha angustifolia L, with a high tolerance to Cr is described. Experiments were carried out to examine its ability to tolerant Cr and its physiological response. The results showed that there was no difference in growth, plant height, and biomass response to external Cr (VI) between the plants exposed to 100 microM Cr (VI) and control (0 microM), while increasing Cr levels to 200-800 microM induced a significant decrease in plant height and biomass, but no significant injury was detected, even for the plants exposed to 800 microM Cr. Chromium induced significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities. Meanwhile, a significantly positive correlation was found between Cr and Mn or Cu in leaves and roots, respectively. The Cr tolerance of the plant appeared to be associated with the enhancement of SOD and POD activities and the improvement in uptake and translocation of the essential microelements.