荧光成像在活体蛋白质研究中的应用

蛋白质的动态特性对蛋白质在细胞内的功能有重要作用.荧光标记、活体内荧光成像技术及显微镜系统的共同进展为人们提供了活细胞内研究蛋白质动态变化及其相互作用的手段.综述了荧光成像技术的进展及其活体内研究蛋白质动态特性中的应用.     

荧光涨落谱的Monte Carlo模拟与应用

荧光涨落谱(Fluorescence Fluctuation Spectroscopy, FFS)技术测量微小区域内扩散粒子的浓度和各种动力学参数, 具有灵敏度高、可以针对活生物体系实时测量的特点, 适用于动态观测特定微小区域中荧光标记生物分子的浓度、质量、体积和结构的变化, 获得活细胞内生物大分子相互作用的信息. 介绍荧光涨落谱实验的Monte Carlo模拟方法, 通过模拟粒子的扩散运动以及荧光激发和收集过程, 预测不同实验体系的荧光涨落谱特征. 利用这种方法可以分析不同实验因素对荧光涨落谱的影响, 验证不同理论计算模型的效果, 为分析、处理复杂生物体系的实验数据提供帮助. 对模拟程序所基于的物理和数学模型进行了介绍, 并结合一些特殊体系中的应用, 对模拟的效果和可靠性进行了多种方式的验证, 得到了与理论分析相符的结果.     

拓展荧光显微镜在细胞生物学实验教学中的应用

荧光显微镜作为一种显微光学观测仪器,在各领域中应用非常广泛。荧光显微镜用于研究细胞内物质的吸收、运输、化学物质的分布及定位等,在植物细胞中可通过荧光染色清晰地观察细胞内的形态及结构。在本科生细胞生物学实验教学中利用荧光显微镜来观察植物组织的自发荧光和次生荧光,掌握荧光显微镜的使用方法、原理及注意事项,同时延伸知识,让学生了解荧光显微镜在生物学领域中的应用。     

一种高选择性苯硫酚荧光探针的研制

设计并合成了一种二硝基苯磺酰基键联2-胺基香豆素的荧光探针分子1,用于水相和活细胞内苯硫酚的检测.探针分子1自身无荧光,加入苯硫酚后,其溶液荧光显著增强,发出黄绿色的强荧光.当加入与探针分子等当量的苯硫酚时,溶液的荧光强度增强了47倍.探针分子1对0~10.00μmol·L~(-1)浓度的苯硫酚呈良好线性响应关系(R2=0.989 9),检测下限为0.46μmol·L~(-1).探针分子1对苯硫酚有较高的选择性和较好的灵敏度.此外,探针分子1成功用于Hep G2细胞内苯硫酚的可视化荧光成像检测.     

一种近红外H2S荧光探针的研制

研制了一种7-硝基-2,1,3-苯并噁二唑-4-醚键联半花菁的近红外区荧光探针分子1,并将其应用于活细胞内内源性和外源性硫化氢的检测.探针分子1由于7-硝基-2,1,3-苯并噁二唑的淬灭作用,自身无荧光;当加入H_2S时,由于H_2S对探针分子1中醚键的选择性硫解作用,醚键断裂,释放出半花菁荧光基团,波长725 nm处荧光强度显著增强;当加入10倍当量的硫氢化钠时,荧光强度增强了8.5倍.探针分子1对水溶液中H_2S浓度的线性浓度响应范围是0.1～100.0μmol·L~(-1)(R~2=0.995 2),检测下限为0.03μmol·L~(-1).探针分子1的激发和发射波长均在近红外区,对H_2S比较敏感,反应时间相对较短,且有较好的选择性.此外,探针分子1被成功用于活细胞内内源性和外源性H_2S的检测.     

心肌细胞原代培养系统的建立及比率荧光技术对细胞内游离钙离子浓度的测定

以Fura-2/AM为指示剂,应用比率荧光测试技术,分别测定心肌细胞和B16细胞单个细胞 内游离钙离子浓度的变化.通过实验,建立了新生大鼠心肌细胞的原代培养系统,并将其应用于 [Ca2 ]i的比率荧光测量.结果显示,心肌细胞在滴加了NE之后,相对荧光强度有明显变化,肯定 了测试系统的有效性,并进一步筛选出适于B16细胞比率荧光测量[Ca2 ]i的各项参数.     

荧光定量检测细胞绿色荧光蛋白技术的建立与应用

绿色荧光蛋白是在生化与分子生物学研究领域中普遍使用的一种基因表达报告系统显色物.对于细胞内绿色荧光蛋白表达水平的定量检测,目前尚缺乏一套准确﹑简便﹑易行的技术.本文以增强型绿色荧光蛋白(EGFP)为报告基因,用分子量为25 ku的线性聚乙烯亚胺介导进入HeLa细胞并表达.利用多功能酶标仪测定细胞裂解液的荧光强度,结果显示:这种新方法测定的荧光强度能很好反映细胞中EGFP的蛋白表达水平,并在短时间内完成对EGFP表达水平的定量检测.该方法简单有效,且不依赖于大型仪器设备,具有较高的准确性与灵敏度,在实际应用中简便可靠,具有推广应用的意义.     

一种检测蛋白质相互作用的双荧光共定位系统

自发荧光蛋白以快捷灵敏、即时检测和无需破坏活细胞的特点在蛋白质相互作用的研究中得到广泛应用.从发光水母Aquoria victoria中分离的绿色荧光蛋白(GFP)和从珊瑚Discosoma sp.中分离的DsRed红色荧光蛋白是自发荧光蛋白的典型代表.本研究开发一种基于GFP和红色荧光蛋白(RFP),并可通过待检测蛋白在细胞内表达的空间位移变化产生共定位的双荧光共定位检测系统.该系统为克服两种荧光蛋白光谱渗漏带来的非特异性结果的干扰,将核仁定位信号(NoLS)和出核信号(ABL)分别连接到RFP和GFP.具有定位信号的GFP和RFP的待检测蛋白对可分别在细胞核内和(或)细胞核外表达,伴随它们的相互作用,荧光共定位的现象会产生在细胞内特定的区域.利用抗凋亡蛋白Bc1-2和Bak-BH3短肽作为蛋白对测试该系统,荧光共定位现象明显,系统灵敏可靠.     

基于矩阵分解的荧光显微序列线粒体检测

利用图像处理技术,检测荧光显微序列中的活性线粒体是生物医学领域重要的研究手段之一.受到荧光显微镜成像技术的限制,序列中每帧图像均包含细胞质阴影和荧光标记的线粒体,具有很低的信噪比,难以满足一般粒子检测算法的要求.为了精确检测活细胞中的线粒体,提出一种基于矩阵分解的荧光显微序列线粒体检测算法,并利用增广拉格朗日乘子法,快速准确地实现该算法,将线粒体从细胞质阴影中有效分离出来,实现线粒体的精确检测.实验结果表明,此方法为活细胞中线粒体的精确检测提供了快速、高效的分析工具.     

三维荧光指纹技术的应用研究进展

搜集了有关三维荧光指纹技术在油种鉴别、水体检测、中药鉴别中应用的研究文献。对三维荧光指纹技术在不同领域中的应用研究进行了综述。结果表明：目前三维荧光指纹技术已被广泛应用在油种鉴别、水体检测领域中。但其在中药鉴别中的应用还不够广泛。三维荧光指纹技术在分析化学的诸多领域起到了重要的作用。具丽广阔的发展前景。     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

Oscillations of cytosolic Ca2+ in pituitary cells due to action potentials

Electrical activity in non-neuronal cells can be induced by altering the membrane potential and eliciting action potentials. For example, hormones, nutrients and neurotransmitters act on excitable endocrine cells. In an attempt to correlate such electrical activity with regulation of cell activation, we report here direct measurements of cytosolic free Ca2+ changes coincident with action potentials. This was achieved by the powerful and novel combination of two complex techniques, the patch clamp and microfluorimetry using fura 2 methodology. Changes in intracellular calcium concentration were monitored in single cells of the pituitary line GH3B6. We show that a single action potential leads to a marked transient increase in cytosolic free calcium. The size of these short-lived maxima is sufficient to evoke secretory activity. The striking kinetic features of these transients enabled us to identify oscillations in intracellular calcium concentration in unperturbed cells resulting from spontaneous action potentials, and hence provide an explanation for basal secretory activity. Somatostatin, an inhibitor of pituitary function, abolishes the spontaneous spiking of free cytosolic Ca2+ which may explain its inhibitory effect on basal prolactin secretion. Our data therefore demonstrate that electrical activity can stimulate Ca2+-dependent functions in excitable non-neuronal cells.     

Intracellular Ca indicator Quin-2 inhibits Ca2+ inflow via Na/Ca exchange in squid axon

Until recently, intracellular free calcium has been amenable to measurement and investigation only in cells large enough to permit either microinjection of a suitable Ca sensor such as a aequorin or arsenazo III or insertion of a Ca-sensitive microelectrode. This constraint on cell size was removed by the development of the fluorescent Ca2+ -sensitive dye Quin-2 and its acetoxymethyl ester, which can be introduced into a wide range of cell types. A major requirement of any intracellular Ca2+ indicator is that it should not disturb intracellular Ca2+ homeostasis and Quin-2 is generally considered to be satisfactory in this respect. We now report that injection of Quin-2 into squid (Loligo forbesi) axons can almost completely abolish one component of Ca2+ entry--intracellular Na+ (Nai)-dependent Ca2+ inflow, which occurs via Na/Ca exchange. Mixtures of Ca and Quin-2 that buffer an ionized Ca2+ at close to physiological concentrations also block Nai-dependent Ca2+ influx but these same mixtures fail to block the extracellular Na+ (Na0)-dependent extrusion of Ca2+, showing that Quin-2 acts specifically on Ca2+ inflow.     

Inhibition of mitosis by an antibody to the mitotic calcium transport system

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.     

用于细胞内自由［Ca~（2＋）］测定的新萤光试剂Fura－2AM的合成、性能及应用研究

用于细胞内自由［Ｃａ２＋］生理作用规律研究的新强萤光试剂Ｆｕｒａ－２ＡＭ已由十七步合成成功。与过去广泛使用的该类试剂相比，其萤光强度高几十倍，对［Ｃａ２＋］亲和力稍弱，对镁和其它二价离子的选择性更好，特别是结合［Ｃａ２＋］后波长发生移动。这些优点使它成为目前最重要的特别是测定单细胞中［Ｃａ２＋］的萤光试剂之一。     

Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration

A rapid, transient rise in the free cytosolic Ca2+ concentration ([Ca2+]i) is one of the earliest events in neutrophil activation and is assumed to be involved in many of the subsequent cellular reactions. Both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space contribute to the rise in [Ca2+]i. In an attempt to assess the relative importance of these pools and the sequences leading to the rise in [Ca2+]i, we have studied the time course of changes in [Ca2+]i after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet-activating factor (PAF) using the Ca2+ indicators quin-2 and fura-2. We observed a time lag of 1-3 s between stimulation and rise in [Ca2+]i. This lag depends on the agonist concentration but is independent of extracellular Ca2+. Thus Ca2+ release from intracellular stores is rate limiting for the rise in [Ca2+]i. After this, cation channels in the plasma membrane (measured with the patch clamp method) are opened. These non-selective channels, which also pass Ca2+, are activated by the initial rise in [Ca2+]i, but by neither fMLP nor inositol 1,4,5-trisphosphate (IP3) directly.     

Use of chlorotetracycline fluorescence to demonstrate Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum of skinned cardiac cells.

It has been proposed that the trans-sarcolemmal influx of Ca2+ occurring during the plateau of the mammalian cardiac action potentials is insufficient in itself to activate the myofilaments, but can trigger a release of Ca2+ from the sarcoplasmic reticulum (SR) which is sufficient for activation. The demonstration of this Ca2+-induced release of Ca2+ relied entirely on experiments in which the tension developed by the myofilaments was used as a sensor of the changes of myoplasmic free Ca2+ concentration ([free Ca2+]) in segments of single cardiac cells from which the sarcolemma had been removed by microdissection (skinned cardiac cells). The small size of these preparations has previously prevented the use of more direct methods for the detection of myoplasmic Ca2+ movements. The present study is a direct demonstration of Ca2+-induced release of Ca2+ from the SR of skinned cardiac cells treated with chlorotetracycline (CTC), a fluorescent chelate probe which enables changes in the amount of Ca2+ bound to a variety of biological membranes or micelles to be monitored. The fluorescence increases when more Ca2+ is bound.     

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.     

Local cytoplasmic calcium gradients in living mitotic cells

Cytoplasmic free calcium has been proposed as a regulator of many microtubule-mediated processes, including mitosis. It has been difficult to test this hypothesis because methods for local measurement of free Ca2+ in the living cell have not been available. We have used the fluorescent calcium indicator dye Quin-2 (methoxyquinoline-1bis(o-aminophenoxy)ethane-N,N,N',N' -tetra acetic acid), which allows such observations to be made by digital processing of fluorescent images from the light microscope. Here we report the application of this technique to the study of local Ca2+ concentrations in mitotic endosperm cells of Haemanthus sp., and show that there is transient increase in free Ca2+ at the mitotic spindle poles during anaphase. This locally high Ca2+ may provide a mechanism for the regional control of microtubules and other cytoskeletal elements during anaphase.