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Dynamic changes of [Ca^(2+)]_i in cerebellar granule cells exposed to pulsed electric fields

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Science China(Life Sciences) 2000年第1期43卷 75-81页

作者：陈雅王彦孙彤张锦珠景向红李瑞午 Department of Cell Biophysics Institute of Biophysics Chinese Academy of Sciences Beijing 100101 China Institute of Acupuncture Chinese Academy of Medicine Beijing 100700 China

Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/am and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field. In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions, the increase of [Ca2+]i was still observable. It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.

关键词： pulsed electric field Ca2+ granule cells fluo-3/am confocal laser scanning microscopy.

High concentration of calcium ions in Golgi apparatus

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Cell Research 1994年第1期4卷 97-108页

作者： XUE SHAOBAI M. RoBERT NICOUD JIE CUI D.J.ARNDT JOVIN(Depariment of Biology, Beijing Normal University, Beijing 100875, China)(Max-Planck-Institute fur Biophysikalische Chemie,Gottingen, Germany) Max-Planck-Institute fSr Biophysikalische Chemie GSttingen Germany

The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6NBD-ceramide, and then the single cells were examined by laser scanning confocal microscopy (LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus. In these cells, the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus. The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle *** calcium free medium, when the plasma membrane of the cells which had been loaded with fluo-3/am were permeated by digitonin, the fluorescence of the Golgi region decreased far less than that of the cytosol. Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.

关键词： intracellular free calcium fluo-3/am Golgi apparatus C6-NBD-ceramide laser scanning confocal microscopy intracellular calcium store

Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

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Cell Research 1995年第2期5卷 165-179页

作者： CUI JIE YAN LI SHAOBAI XUE(Biology Department, Beijing Normal University, Beijing100875, China.) (Meridian Instruments (H K. (LTD), Unit 806, 8/F.,Cheung TAT Centre, 18 Cheung Lee Street, Shai Wan,Hong Kong.) Meridian Instruments (H.K. (LTD)) Unit 806 8/F. Cheung TAT Centre 18 Cheung Lee Street Shai Wan Hong Kong.

Using laser scanning confocal microscopy, we have found that the in cells loaded with fluo-3/am, highest intracellular Ca(2+) in the perinuclear region is associated with the Golgi apparatus. The spatiotemporal subcellu lar distribution of Ca(2+) in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated. PDGF, which releases Ca(2+) from intracellular stores by inositol(1, 4, 5)-trisphosphate pathway,produced a biphasic transient rise in intracellular *** initial rise was resulted from a direct release of calcium from the Golgi apparatus. Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin, an inhibitor of the Ca(2+) transport ATPase of intracellular calcium store. Permeablizing the plasma membrane by 10 μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store. These results suggest that the Golgi apparatus plays a role in Ca(2+) regulation in signal transduction.

关键词： Golgi apparatus intracellular calcium store fluo-3/am laser scanning confocal microscopy PDGF thapsigargin

Effect of melatonin on the spatial and temporal changes of [Ca^(2+) ]i in single living cells of cortical neurons by laser scanning confocal microscopy

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Chinese Medical Journal 2000年第6期113卷 78-82页

作者：张庆柱张均田 China 100050

Objective To examine the effects of melatonin on the dynamic changes in the concentration of intracellular free Ca 2+ ([Ca 2+ ]i) in single intact cultured cortical neurons isolated from fetal rats, in order to explore the possible antiaging mechanisms of melatonin (MT) Methods Using the highly fluorescent Ca 2+ sensitive indicator fluo 3/am, cortical neurons cultured in a 35?mm Tissue Culture Dish were in incubated for 45?min at room temperature with 5?μmol/L fluo 3/am, resulting in proper intracellular dye concentration to provide adequate signal strength for detection and excellent Laser Scanning Confocal Microscopy (LSCM) imaging of [Ca 2+ ]i while not disturbing normal intracellular physiology The changes in fluorescent intensity were monitored by LSCM Results Bay K8644 (10 6 ?mol/L), KCl (20 ?mmol/L), sodium L glutamate (Glu, 50?μmol/L) caused a rapid increase of [Ca 2+ ]i in cortical neurons, and this increase could be significantly attenuated by 10 6 and 10 7 mol/L MT Conclusions MT could antagonize the extracellular Ca 2+ influx, reduce Ca 2+ overload, and have a protective effect on neurons This may be one of the important antiaging mechanisms of MT

关键词： melatonin calcium laser scanning confocal microscopy fluo-3/am cerebral cortex neurons primary cell cultures aging

低温处理对冬、春小麦细胞Ca^(2+)时空变化的影响

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Acta Botanica Sinica 2001年第12期43卷 1218-1223页

作者：刘炜孙德兰王红简令成尚忠林王学臣赵可夫中国科学院植物研究所北京100093 中国农业大学生物学院农业部植物生理生化重点开放实验室北京100094 山东师范大学逆境植物研究所济南250014

用fluo 3/am染色 ,通过激光扫描共聚焦显微镜 (LSCM)方法 ,对静息态及连续降温条件下不同抗寒性小麦(TriticumaestivumL .)叶肉细胞原生质体 [Ca2 + ]cyt (thefreeCa2 + concentrationinthecytoplasm)的时空变化进行了比较。结果表明 ,... 详细信息

用fluo 3/am染色 ,通过激光扫描共聚焦显微镜 (LSCM)方法 ,对静息态及连续降温条件下不同抗寒性小麦(TriticumaestivumL .)叶肉细胞原生质体 [Ca2 + ]cyt (thefreeCa2 + concentrationinthecytoplasm)的时空变化进行了比较。结果表明 ,静息态下小麦原生质体整体荧光强度基本不变 ,暗示 [Ca2 + ]cyt能维持在一稳定水平 ;同时 ,不同品种小麦间也显示了 [Ca2 + ]cyt水平荧光强度的不同。温度由 15℃连续降至约 2℃时 ,抗寒冬小麦 [Ca2 + ]cyt出现升高后的回复 ,2℃之后逐渐升高 ;冷敏感春小麦则无此回复过程 ,而是一直升高到最大值。推测这一不同的动态变化最终决定了植物在低温下产生冷适应的不同能力。这进一步为“Ca2 + 是低温下生理信号的传导者”

关键词： fluo-3/am 小麦原生质体静息态 Ca^2+ 时空变化钙

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丹参悬浮培养细胞原生质体的制备和活力检测

生物工程学报 2014年第10期30卷 1612-1621页

作者：朱楠刘俊张馨宇董娟娥西北农林科技大学生命科学学院陕西杨凌712100

对丹参悬浮培养细胞原生质体制备条件进行了研究,并利用FDA染色和钙离子荧光探针fluo-3/am装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为:悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果... 详细信息

对丹参悬浮培养细胞原生质体制备条件进行了研究,并利用FDA染色和钙离子荧光探针fluo-3/am装载对制备得到的原生质体的活力和功能进行了检测。丹参悬浮培养细胞原生质体的制备条件为:悬浮培养细胞酶解的适宜酶液组合为纤维素酶1.5%、果胶酶0.3%和离析酶0.5%;适宜的甘露醇浓度为0.4 mol/L;酶解时间为12 h;在600 r/min转速下离心5 min收集,纯化得到原生质体,其产量为1.1×106/g FW,FDA检测显示其活力为95%以上,荧光探针fluo-3/am可成功装载到原生质体中。

关键词：丹参原生质体激光共聚焦 FDA fluo-3/am

共聚焦激光扫描显微镜技术在研究大鼠海马神经元胞内钙超载中的应用

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复旦学报（医学版） 2005年第1期32卷 98-100,104,F003页

作者：黄娅林赵鹏程介士复旦大学上海医学院医学神经生物学国家重点实验室上海200032

目的观察抑制性氨基酸牛磺酸对兴奋性氨基酸谷氨酸所致胞内钙超载的影响。方法选择fluo-3／ am对培养的海马神经元进行着色后,用共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)实时扫描,动态显示海马神经元二维图像... 详细信息

目的观察抑制性氨基酸牛磺酸对兴奋性氨基酸谷氨酸所致胞内钙超载的影响。方法选择fluo-3／ am对培养的海马神经元进行着色后,用共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)实时扫描,动态显示海马神经元二维图像,以计算机相应软件对胞内钙的荧光强度变化进行分析统计。结果在 0.5 mmol／L谷氨酸作用下,胞内钙荧光强度迅速升高(P3 mmol／L牛磺酸共同作用于海马神经元时,胞内钙荧光强度明显降低(P<0.001);灌流液中去除牛磺酸后,胞内钙荧光强度上升(P <0.001)。结论抑制性氨基酸牛磺酸可抑制由兴奋性氨基酸谷氨酸所致神经元的胞内钙超载;CLSM以其独特的优势,在高清晰度地显示海马神经元形态的同时,也可动态地观察活细胞在不同环境下胞内钙的快速变化以及定量分析。该技术在动态观察和二维图像的高分辨率上比传统测钙方法有更大的优越性。

关键词：钙超载海马神经元共聚焦激光扫描显微镜牛磺酸谷氨酸 CLSM 大鼠海马荧光强度活细胞 fluo-3/am

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西红花苷对培养的牛内皮细胞内钙的调节作用

中国药科大学学报 2002年第5期33卷 445-447页

作者：绪广林钱之玉任萱中国药科大学药理学教研室南京210009

目的观察西红花苷对不同刺激因子5-羟色胺（5-HT）、组胺（His）、过氧化氢（H2O2）所致内皮细胞内钙升高的影响。方法采用fluo-3/am荧光染色剂负载细胞,在激光共聚焦扫描显微镜上测定培养的单个牛内皮细胞内游离钙的浓度。结果在含... 详细信息

目的观察西红花苷对不同刺激因子5-羟色胺（5-HT）、组胺（His）、过氧化氢（H2O2）所致内皮细胞内钙升高的影响。方法采用fluo-3/am荧光染色剂负载细胞,在激光共聚焦扫描显微镜上测定培养的单个牛内皮细胞内游离钙的浓度。结果在含钙的Hank’s液中,5-经色胺、组胺、过氧化氢能明显升高静息状态的细胞内钙,增幅分别为280％、320％、285％。西红花苷1、10μmol/L对5-羟色胺、组胺、过氧化氢引起的钙增高有显著的抑制作用,并呈一定的剂量依赖性。0.1μmol/L西红花苷对细胞内钙升高无显著影响。结论西红花苷抗动脉粥样硬化、高血压及炎症作用可能与其内皮细胞保护作用有关。

关键词：西红花苷 fluo-3/am 激光共聚焦扫描显微镜内皮细胞细胞内钙心血管病

西红花苷对牛主动脉平滑肌细胞内钙离子浓度的影响(英文)

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药学学报 2004年第10期39卷 778-781页

作者：何书英钱之玉唐富天中国药科大学药理教研室江苏南京210009

目的研究西红花苷对血管平滑肌细胞内钙离子浓度的影响。方法以fluo-3/am作为Ca2+荧光探针,采用激光扫描共聚焦显微镜观察牛主动脉平滑肌细胞内钙离子浓度的变化。结果无论细胞外有无钙离子,西红花苷(1×10～8,1×10～7,1×10～6mol·... 详细信息

目的研究西红花苷对血管平滑肌细胞内钙离子浓度的影响。方法以fluo-3/am作为Ca2+荧光探针,采用激光扫描共聚焦显微镜观察牛主动脉平滑肌细胞内钙离子浓度的变化。结果无论细胞外有无钙离子,西红花苷(1×10～8,1×10～7,1×10～6mol·L-1)均能明显抑制1×10～2mol·L-1H2O2引起的细胞内钙离子浓度的升高,其抑制率含钙条件下分别为34.1％,57.1％和74.3％(P32.1％和50.0％(P3导致雷洛丁敏感钙池的释放,其抑制率分别为27.8％,27.8％和50.0％(P<0.01)。结论西红花苷能抑制胞外钙离子的内流及内质网上钙离子的释放。

关键词：西红花苷激光共聚焦显微镜平滑肌细胞 fluo-3/am 钙离子