Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Fluorescent stains for quantification of DNA by confocal laser scanning microscopy in 3-D

Confocal microscopy requires the use of fluorophores to visualize structures of interest within a specimen. To perform reliable measurements of the intensity of fluorescence, the stain should be specific, penetrate well into tissue sections, and bind stoichiometrically. Furthermore, emission must be linear with respect to DNA content and brightness, and fluorescence should be stable. Confocal microscopy is used to determine DNA ploidy and to analyze texture of nuclei, which is accomplished in three dimensions, because nuclei can be measured within the original tissue context. For this purpose the sample must be stained with a DNA binding fluorophore with the properties described above. Stains with different properties have been developed for different applications. We review here the advantages and disadvantages of these different stains for analyzing DNA ploidy and nuclear texture using three-dimensional microscopy. We conclude that SYBR green I and TO-PRO-3 are the most suitable stains for this purpose at present.     

双光子及共聚焦激光扫描显微术研究天花粉蛋白对人绒癌细胞的作用机制

天花粉蛋白(trichosanthin, TCS)是从中草药栝楼根中提取的一种核糖体失活蛋白,具有抗肿瘤和抗HIV功能.应用双光子及共聚焦激光扫描显微术结合特异性荧光探针Hoechst 33342、2′,7′-二氯荧光黄双乙酸酯 (DCFH-DA)、Indo-1和Fluo 3-AM,首次同时观察了TCS诱导人绒癌细胞（JAR细胞）凋亡过程中活性氧自由基（ROS）和细胞内钙离子浓度（［Ca²⁺］_i）的变化,实验结果表明TCS引起的［Ca²⁺］_i升高和ROS形成参与了TCS诱导的JAR细胞凋亡,并且ROS形成和［Ca²⁺］_i升高有关.共聚焦激光扫描显微术的研究结果表明,［Ca²⁺］_i升高不是导致ROS形成的主要原因,TCS诱导产生的ROS可能是通过TCS与JAR细胞膜表面受体作用介导的.     

活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。     

酪丝亮肽荧光标记物的合成及其在肿瘤治疗靶点研究中的应用

酪丝亮肽是一种具有抗肿瘤活性的小分子三肽,它可以诱导造成肝癌细胞发生凋亡坏死,从而杀伤肿瘤细胞,但是酪丝亮肽在肝癌细胞的亚细胞定位尚不十分明确．为了达到对酪丝亮肽进行示踪进而观察其亚细胞定位的目的,使用荧光物质（5（6）－羧基叫甲基罗丹明琥珀酰业胺酯,5（6）－TAMRASE）对酪丝亮肽进行丫标记,应用非变性聚丙烯酰胺凝胶电泳、毛细管电泳和荧光分光光度法对标记酪丝亮肽进行纯化和鉴定．并在激光扫描共聚焦显微镜下观察了荧光标记酪丝亮肽在人肝癌BEL－7402细胞中的分布．结果显示,合成的酪丝亮肽荧光标记物性质稳定,标记的酪丝亮肽在人肝癌BEL－7402细胞的胞浆中呈聚集分布．     

Effects of phorbol esters on endothelial cell microfilaments: Laser scanning confocal microscopy and quantitative morphometry of dose dependent changes

Phorbol esters are known to alter microfilaments but it is not clear if the changes correspond to modulation of the phosphoinositide turnover/protein kinase C system. The novel technique of laser scanning confocal epifluorescence was used to study fiber orientation in phorbol ester treated cells. We treated endothelial cells with control agents and agents known to stimulate protein kinase C: 4 alpha-phorbol, phorbol 12-myristate 13-acetate (PMA), phorbol dibutyrate (PDB), or lipopolysaccharide. After incubation with the test agents, the endothelial cell microfilaments were stained with rhodamine pholloidin and viewed by conventional epifluorescence and by laser scanning confocal epifluorescence microscopy. The images obtained by the confocal microscopy corresponded to a thin optical section through the cells, 300 nm or more in thickness. The microfilaments extended predominantly in the plane of focus. After exposure of the cells to phorbol esters, the stress fibers became more nearly parallel in arrangement or were shortened, but remained in the plane of focus. The modification of microfilaments in response to phorbol esters was quantitated by a single blind analysis. In order to compare the morphological changes with a biochemical action of the phorbol esters, we measured phosphoinositide turnover. The dose-dependence of morphological changes was compared and contrasted to the dose-dependent effect of phorbol esters on bradykinin-stimulated phosphoinositide turnover. PMA had about the same EC50 (1-5 nM) for both biochemical and morphological processes. PDB was less potent in inducing the disruption of microfilament structure than in inhibiting phosphoinositide turnover. Lipopolysaccharide was ineffective in inducing a morphological change under these conditions. A simple activation of protein kinase C is insufficient to explain the dose-dependent effects of phorbol esters. Thus a morphometric analysis can help distinguish the potency of cytoskeleton modulators.     

Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy

In the present study a new luminescent dye 3‐N‐(2‐pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3‐aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.     

非培养细胞的贴壁方法及细胞内Ca^2＋观测

本文介绍非培养细胞贴壁的简易方法和细胞内Ｃａ＾２＋观察。以ｆｌｕｏ－３／ＡＭ进行染色,在３７℃孵育箱内孵育６０分钟,用激光共聚焦显微镜监测细胞内Ｃａ＾２＋的荧光信号。结果表明本方法可成功地应用于共聚焦显微镜测定细胞的研究。     

钙结合蛋白CIB1在293T细胞中的表达及亚细胞定位研究

分析和比对钙结合蛋白CIB1在人类、大鼠、小鼠中氨基酸序列差异,并研究CIB1蛋白在293T细胞中的表达及亚细胞定位情况。通过Western Blot分析发现293T细胞本身几乎检测不到CIB1的表达。进一步采用CIB1外源性质粒转染,通过免疫荧光分析实验,在激光共聚焦显微镜下观察转染后不同时间293T细胞中CIB1的表达情况及亚细胞定位变化。实验结果表明,随着转染时间的增加,CIB1在293T细胞中的表达有逐渐增强的趋势,并且发生从细胞核向细胞质的转位现象。该实验结果对了解CIB1亚细胞定位变化及功能研究具有重要参考价值。     

增龄引起复制性衰老细胞胞内钙信号转导的变化

为了观察血管紧张素Ⅱ (AngⅡ )引起复制性衰老细胞胞内游离钙的变化以及衰老对其的影响 ,初步阐明衰老引起胞内游离钙变化的机制 .选用人胚肺二倍体成纤维细胞WI 38细胞株 ,利用逆转录PCR(RT PCR)技术及Northern杂交技术检测衰老细胞血管紧张素Ⅱ 1型受体 (AT1R)、血管紧张素Ⅱ 2型受体 (AT2R)mRNA水平的表达 ;利用激光共聚焦显微成像技术 (LSCM )观察WI 38细胞在AngⅡ刺激 ,Valsartan阻断条件下细胞内钙离子荧光强度的改变 .AngⅡ通过AT1受体介导增加WI 38细胞内游离钙的水平 ,并随着WI 38细胞传代增加至衰老状态 ,AT2受体高表达 ,AT1受体介导的钙离子信号转导的活性逐渐降低 .提示WI 38细胞衰老过程中钙离子信号的转导活性降低 ,并且AT1R和AT2R在血管紧张素Ⅱ介导的细胞内钙信号活性中具有不同的作用与机制 .为探讨WI 38衰老细胞内钙信号变化机制提供了实验依据     

荧光成像技术及其在生命科学中的应用

近年来,荧光成像技术发展迅速,其成像系统通常为目前最先进的分析检测仪器之一的激光共聚焦显微镜,荧光探针是荧光成像技术的核心之一。作为新兴光学成像技术,荧光成像技术在生命科学领域中应用广泛,可用于蛋白质及金属离子检测,肿瘤疾病的诊断,并为药物新剂型的研究提供了新思路。     

Glomerulus development in the absence of a set of mitral-like neurons in the insect olfactory lobe

Mitral cells are the first neurons in the mammalian olfactory bulb to synapse with olfactory receptor axons during glomerulus development, and in an invertebrate, the moth Manduca sexta, mitral-like neurons overlap very early with olfactory receptor axons as they begin to form protoglomeruli. The possibility for early interaction between receptor neurons and mitral-like neurons led us to ask whether such an interaction plays an essential role in glomerulus development. In the current study in the moth, we surgically removed a major class of these mitral-like neurons before glomeruli began to form and asked: (a) Is the formation of the array of olfactory glomeruli triggered by an interaction of the first-arriving receptor axons with the dendrites of mitral-like neurons? (b) At the level of individual glomeruli, must the mitral-like dendrites be in place either to maintain receptor axons in a glomerular arrangement, or to guide later-growing dendrites of other types into the developing glomeruli? Our results indicate that even without the participation of this group of mitral-like neurons, the array of sexually isomorphic ordinary glomeruli forms and the basic substructure of individual glomeruli develops apparently normally. We conclude that the mitral-like neurons in Manduca are not essential for the formation of ordinary olfactory glomeruli during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 41–52, 1998     

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

过氧化氢在水杨酸诱导的蚕豆气孔关闭中的作用

许多植物病原菌可通过气孔进入叶片组织,因此减小气孔开度有利于提高植物的抗性。我们通过表皮条分析和激光扫描共聚显微镜得到的证据表明在保卫细胞中过氧化氢可能是水杨酸信号的中间环节。SA可以浓度依赖的方式诱导气孔关闭（图1A）,H2O2也有类似的作用（图1B）。100μmol/L的水杨酸诱导的气孔关闭作用可明显地被20U/ml的过氧化氢酶或10μmol/L的Vc逆转,但CAT和Vc单独处理时诱导气孔开放的作用很微弱。单细胞中基于荧光探针DCFH的时间进程实验表明直接外加（图版I）或显微注射100μmol/L的SA均可诱导保卫细胞中H2O2产生,但以显微注射双蒸水作为对照时对DCFH荧光无影响（图版II）。这些结果暗示了植物被病原菌感染时可能通过产生H2O2导致气孔关闭而阻止病原菌继续通过气孔侵入。     

Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.     

变应性鼻炎鼻分泌物中细胞内DNA和RNA代谢的观测

本文介绍应变性鼻炎鼻分泌物中细胞内DNA和RNA代谢变化的观测方法。以鼻分泌物中的多种炎性细胞为实验标本,经细胞贴壁、0.1%吖橙染色标记,37℃孵育15min;采用激光扫描共聚焦显微镜对细胞内DNA,RNA形态和含量进行观测。结果表明：本方法应用于变应性鼻炎鼻分泌物细胞内DNA、RNA代谢的研究是切实可行的。     

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.     

RAB5A基因对肺腺癌细胞微丝的影响

为研究RAB5A基因对肺腺癌细胞中微丝的影响,通过FITC标记的鬼笔环肽对AGZY83-a细胞骨架中的微丝特异染色,利用共聚焦激光扫描显微镜发现RAB5A过表达后微丝束变密。经Superarray肿瘤转移相关基因微芯片分析RAB5A对肿瘤转移相关基因的表达影响．发现了3个与细胞骨架调节相关基因表达发生变化,NM23H1与Rac1的表达受到抑制．同时S100A4的表达增加。以前有研究认为S100A4基因可抑制NM23H1基因表达,为验证NM23H1基因的表达降低是否由于S100肖4表达增高所致,利用RNAi沉默AGZY83-a细胞中S100A4基因的表达,发现NM23H1基因表达增高,由此推断RAB5A基因可能通过上调S100A4基因表达来抑制NM23H1基因表达。