细胞内pH值测定方法的研究进展

很多细胞的活动都对pH的变化十分敏感。pH值的有效控制对维持正常的细胞生理活动十分重要。如何有效监测细胞内pH值是很多细胞生物学的重要研究内容之一,如在研究细胞内转运蛋白、Ca2+离子等的变化活动时均需要测定细胞内的pH值,其相关的研究已有100多年的历史。本文将介绍目前几种细胞内pH值的主要测定方法,包括:弱酸弱碱分布法、核磁共振法、微电极法、荧光探针法等;每一种方法将从相关方法、技术的原理、特点、应用、局限性和注意事项等着手,将各个方法的优缺点进行横向的比较。本文还将重点探讨免疫探针法的最新进展,将报道一种最新的基于碳纳米点技术和荧光染料结合的pH定量测定计,还将介绍SNARF的两种最新衍生染料SNARF-F和SNARF-Cl的性能及其应用。     

pH敏感型荧光蛋白及其在植物细胞生物学中的应用

pH敏感型荧光蛋白, 即pHluorin, 是荧光强度及光谱特征随环境pH值的变化而改变的一类荧光蛋白。人们通过对密码子使用偏好和特定剪切位点的修饰, 已使pHluorin及其衍生物成功地在动物、植物和真菌细胞中正常表达, 为测量细胞内微环境pH值的变化, 并研究活细胞内依赖或导致pH变化的生理过程提供了有力工具。该文总结了目前已报道的pH敏感型荧光蛋白的种类及特性, 并对其在细胞生物学, 特别是植物细胞生物学中的应用进行了详细介绍。随着报告基因技术及检测方法的不断改进, pHluorin将在植物科学领域发挥更大的作用。     

监测细胞内氧化还原代谢状态的遗传编码荧光探针

原位、高时空分辨地检测细胞内氧化还原代谢状态是生命科学研究的一个瓶颈问题和迫切需求.然而,依赖细胞裂解、酶学、色谱、质谱等传统生化分析方法难以实时监测细胞内氧化还原代谢变化,更难以应用于高通量药物筛选.基于荧光蛋白的探针成像是近年来生命科学和医学领域迅速发展的一种分析检测技术.由于这些荧光探针实现了在活细胞内实时、动态地监测生物学过程,从而革命性地改变了生命科学研究.相对于化学小分子荧光探针,遗传编码的荧光蛋白探针在精确定位亚细胞结构、消除人为干扰以及活体应用方面,存在显著的优势.近年来,科学家针对细胞内重要的氧化还原代谢物,发明了多种多样的遗传编码荧光探针,实现了在单细胞、亚细胞甚至活体内对氧化还原代谢状态的特异性检测和成像,大大推动了相关研究领域的发展.本文将以细胞内两对关键的氧化还原代谢分子NADH/NAD~+和NADPH/NADP~+为例,重点介绍相关荧光探针的设计、性质、应用以及使用注意事项,以方便研究者更好地了解和使用相关技术.     

天麻食菌过程中蜜环菌活力的变化及几种酶的组织化学定位

应用吖啶橙活体染色法,借助于荧光显微镜观察了侵染天麻球茎的蜜环菌活力的变化。发现初染菌丝发射出具活力的绿色荧光,衰老菌丝为黄色荧光,而残缺菌丝及丛状体则发射出失活的橙红色荧光。用吖啶橙诱导荧光法及在 pH2.2的条件下以固绿染色的方法,证实了大型细胞内含有丰富的 RNA 和总蛋白质。酸性磷酸酶主要分布在含菌丝的皮层细胞、大型细胞除细胞壁外,细胞内只有较少的反应沉淀物。酯酶在含菌丝的皮层细胞内显示了非常明显的活性,而在大型细胞的丛状体及崩解核内也显示了明显的活性分布。ATP 酶,多酚氧化酶与过氧化物酶在含菌丝的皮层细胞及大型细胞内均显示了明显的活性。     

植物体内Ca2+,pH,ROS活体荧光探针成像研究进展

细胞内信号分子Ca2+与活性氧(ROS)以及细胞pH,是参与细胞多种生理和发育过程调节的关键因子。这些信号分子或细胞化学势之所以在如此众多方面起作用,是因为它们在生物体内从细胞到器官水平上、从数秒到数小时一直是在时空动态变化着的。恰到好处的是,荧光素感受器可以稳定地对细胞内Ca2+,pH与活性氧(ROS)活体原位实时定量。可视化的荧光细胞探针可分为两类:(a)直接染色;(b)基于绿色荧光蛋由基因编码的感受器。绿色荧光蛋白探针可在亚细胞水平上对目标蛋白准确定位,为得到细胞信号高分辨图提供可能。     

华蟾素对MDA - MB -231细胞线粒体膜电位及活性氧的影响

目的:研究华蟾素诱导乳腺癌MDA - MB - 231细胞凋亡过程中,细胞内活性氧(ROS)及线粒体膜电位(△Ψm)的变化,探讨华蟾素对乳腺癌细胞的作用机制.方法:用不同浓度的华蟾素作用于MDA - MB - 231细胞24h后,分别用荧光探针罗丹明123和荧光探针DCFH-DA进行荧光染色,用流式细胞仪检测细胞内线粒体膜电位和活性氧的变化.结果:不同浓度的华蟾素作用于MDA - MB - 231细胞后,随着药物浓度的增加(0、12.5、25、37.5、50μg/ml),细胞内的ROS水平显著升高,荧光强度从3 609±24上升为6 263±35;同时,线粒体膜电位(△Ψm)显著下降,荧光强度从242±6降低到173±4.结论:华蟾素作用细胞后,使得细胞内活性氧水平显著升高,同时,线粒体膜电位显著下降,推测华蟾素对MDA - MB - 231细胞通过线粒体途径诱发细胞凋亡.     

细胞结构动力学——实时调控细胞力学的因素分析

机械力普遍存在于活细胞的生命活动中,而细胞内力学活动必须依赖骨架结构传递,这种独特的力学形式被称为细胞结构力学.单位时间内细胞结构力学变化受多因素调控,如外力、渗透压、动力分子、张力敏感性离子通道、胞内力学感受器及骨架组装等,构成了细胞结构动力学研究的重要内容.基于荧光共振能量转移(FRET)原理开发的荧光张力探针能整合到细胞骨架内,将细胞结构力学变化转化为光学信号,可能带来细胞力学研究的革命.随着细胞结构动力学研究内容的不断深入,特别是太空时代细胞力学稳态的打破,细胞结构动力学将在生命及医学研究领域显露出越来越重要的地位.     

pH改变对心肌细胞内Ca~(2 )浓度和细胞长度的影响

目的 :探讨细胞内 pH(pHi)改变对心肌细胞内Ca2 浓度 ([Ca2 ]i)和细胞长度的影响。方法 :心肌细胞内分别灌注 2 0mmol/L丙酸钠和 15mmol/LNH4Cl,建立细胞内酸碱中毒模型。荧光指示剂indo 1和SNARF 1载入大鼠心肌细胞内 ,用荧光显微镜同时测定心肌 [Ca2 ]i、pHi 和细胞长度。结果 :细胞内酸中毒早期 ,收缩期和舒张期[Ca2 ]i 轻度增加 ,细胞缩短 (CS)降低 ,细胞长度增加 ,心肌纤维对Ca2 的敏感性和CS/ [Ca2 ]i 降低 (P <0 .0 1) ;碱中毒时 ,收缩期和舒张期 [Ca2 ]i 均较对照组降低 ,CS增加 ,细胞长度变短 ,心肌纤维对Ca2 的敏感性和CS/[Ca2 ]i 增加 (P <0 .0 1)。结论 :酸中毒早期 [Ca2 ]i 和细胞长度增加 ,碱中毒时 [Ca2 ]i和细胞长度降低。酸、碱中毒对Ca2 敏感性的影响并非线性关系 ,即单位 pHi变化时酸中毒对敏感性的影响较碱中毒小     

小鼠巨噬细胞内游离钙离子变化介导的吞噬作用

为评估小鼠巨噬细胞吞噬死亡细胞时胞质内游离钙离子的变化。实验使用F luo-3标记巨噬细胞内钙离子和碘化丙碇对死亡细胞核染色,观察吞噬过程中细胞内钙离子的变化和显示巨噬细胞的吞噬功能,检测含死亡细胞的巨噬细胞内荧光密度图像。利用激光扫描共聚焦显微镜检测钙离子的释放。在缺钙的溶液中,可见巨噬细胞接触死细胞时细胞内钙离子快速地聚集和增高。在吞噬体形成时,巨噬细胞内钙离子上升到较高的水平。快速上升后,当吞噬小泡消化时,细胞内游离钙下降,随后钙离子恢复到低水平。研究显示伴随着吞噬小泡中红色荧光的死细胞的出现和消失,巨噬细胞内出现一系列钙离子变化的图像。提示巨噬细胞内钙离子改变在细胞吞噬作用中具有一定的作用。     

pH改变对心肌细胞内Ca2+浓度和细胞长度的影响

目的：探讨细胞内pH(pHi)改变对心肌细胞内Ca^2 浓度（[Ca^2 ]i)和细胞长度的影响。方法：心肌细胞内分别灌注20mmol/L丙酸钠和15mmol/L NH4Cl ,建立细胞内酸碱中毒模型。荧光指示剂indo-1和SNARF－1载入大鼠心肌细胞内,用荧光显微镜同时测定心肌[Ca^2 ]i、pHi和细胞长度。结果：细胞内酸中毒早期,收缩期和舒张期[Ca^2 ]i轻度增加,细胞缩短（CS）降低,细胞长度增加,心肌纤维对Ca^2 的敏感性和CS／[Ca^2 ]i降低（P＜0．01）;碱中毒时,收缩期和舒张期[Ca^2 ]i均较对照组降低,CS增加,细胞长度变短,心肌纤维对Ca^2 的敏感性和CS／[Ca^2 ]i增加（P＜0．01）。结论：酸中毒早期[Ca^2 ]i和细胞长度增加,碱中毒时[Ca^2 ]i和细胞长度降低。酸、碱中毒对Ca^2＋敏感性的影响并非线性关系,即单位pHi变化时酸中毒对敏感性的影响较碱中毒小。     

Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine

In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pKa of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load.     

Quin-2 and fura-2 measure calcium differently

Several fluorescent probes have been used in the past to monitor and to measure intracellular calcium and calcium fluxes. The most widely used of these probes are those developed by Tsien. We address the markedly different values obtained when comparing Quin-2 (the original probe) with Fura-2 (a second-generation probe). In most cases the values for intracellular calcium have been considered to be interchangeable for the different probes. Using several different hematopoietic cell lines we show that in no case do the two probes yield equivalent values.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells.

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and 2-p-toluidinyl-6-naphthalene sulfonate (TNS) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Determination of intracellular pH using sensitive, clickable fluorescent probes

We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence, high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indicated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Additionally, its pK(a) value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring of intracellular [H(+)] levels. The easily accessible terminal alkyne/azido function groups of these probes offer the possibility of rapidly constructing sensor molecule libraries using 'click' chemistry.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and $\text{2-p-}\text{toluidinyl-6-naphthalene}$ sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

钙荧光探剂的研究及其在生命科学中的应用

钙荧光探剂测量活细胞胞浆游离Ｃａ２＋浓度的方法在钙研究中已成为一种越来越重要的技术。特别是由于新的一代荧光探剂的合成和激光共聚焦显微镜的发展,使其应用更加广泛。由于国内使用这种技术的实验室逐渐增多,本文将系统介绍钙荧光探剂的发展、测量原理和方法、新的常用钙荧光探剂的比较及其在生命科学中的应用。     

Receptor-mediated targeting of fluorescent probes in living cells

A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties.     

Video fluorescence microscopy as a tool for the study of cellular heterogeneity in epithelia

The use of functional fluorescent dyes has allowed us to monitor intracellular pH in individually identified cells in renal epithelia. Using video microscopy we simultaneously measured the change in intracellular pH in several contiguous cells in response to various maneuvers. The video equipment included a silicon intensified target camera, a VHS videocassette recorder, a high resolution monochrome monitor, a video photometric analyzer and a 2-channel chart recorder. This equipment had a spatial resolution of 1 micron by light microscopy and a response time of less than 200 ms; it allowed us to perform double fluorescent labeling and obtain reliable measurements of intracellular pH, independent of gain, regardless of the location of the image on the screen. Using this video system we have shown that there is substantial heterogeneity in activity of H+/HCO3- transport pathways among adjacent cells in a monolayer of cells cultured from the rat renal inner medullary collecting duct. In isolated perfused rabbit renal cortical collecting ducts, video microscopy allowed us to show that there are two different types of intercalated cells: one that exhibits apical Cl-/HCO3- exchange and one that does not. Both show alkaline intracellular pH with respect to non-acid-base transporting epithelia. Video microscopy has several advantages over conventional microspectrophotometry. It provides rapid data acquisition along with increased sensitivity and the capacity for some subcellular analyses. One is able to analyze several individually identified cells during an experimental maneuver. The present video system was assembled for less than $15,000 and permits a more complete analysis of an epithelium than either single-cell photometry or spectrophotometric analysis of thousands of cells in suspension or monolayers.     

The slowing of Ca2+ signals by Ca2+ indicators in cardiac muscle.

The use of high-affinity fluorescent probes for monitoring intracellular free Ca2+ in cardiac muscle is now widespread. We have investigated the consequences of introducing intracellular buffers with the properties of Fura-2 or Indo-1 on the action potential, Ca2+ transient and contractile activity of the myocardium. Our theoretical results suggest that, at the high intracellular concentrations of these fluorescent probes used on occasion to improve the signal-to-noise ratio of the emitted fluorescence, modulation of action potential profile and attenuation of the amplitudes of the Ca2+ transient and contraction can occur, together with subtle changes in the kinetics of these events.