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。     

用荧光漂白恢复技术研究竹红菌乙素在小鼠肝癌细胞内的扩散过程

利用竹红菌乙素自身的荧光特征,在ＦＰＲ装置上直接测定了乙素在ＡＨ细胞内的侧向扩散系数和荧光漂白的恢复率。实验结果表明乙素在ＡＨ细胞内的扩散系数Ｄ＝３．２×１０＾－９ｃｍ＾２／ｓ＾－１荧光漂白恢复比率Ｒ＝９７．８％,上述实验说明乙素在细胞膜内与生物大分子之间没有形成共价键形式的结合状态。     

凝血酶引致的血小板内钙,镁离子浓度及分布状态的变化

我们使用荧光探针ｆｕｒａ２、ｍａｇ－ｆｕｒａ２和ｆｌｕｏ３测定了凝血酶引致的血小板凝聚过程中细胞内钙、镁离子浓度的变化及分布状态。在０．５Ｕ／ｍｌ凝血酶作用下,血小板细胞内钙离子浓度呈双时相变化。血小板细胞群中细胞内钙离子浓度呈正态分布。伴随血小板凝聚时细胞内钙离子浓度增加,血小板细胞内游离镁离子浓度也明显增加,提示镁离子在血小板凝聚中有重要的作用。     

脑啡肽对小鼠神经母细胞瘤细胞脂褐素影响的实验研究

本文应用小鼠神经母细胞瘤细胞Ａ２（ＮＢ－Ａ２）无血清培养建立的神经细胞老化实验研究模型,以显微荧光分光光度术测定单个细胞内脂褐素荧光值作为细胞老化指标,观察了脑啡肽（ＬＥ）对ＮＢＡ２细胞内脂褐素荧光值的影响。结果表明ＬＥ可显著降低细胞内的脂褐素荧光值（Ｐ〈０．０１）。提示ＬＥ可延缓ＮＢＡ２细胞的老化进程。     

对果蝇S期细胞内组蛋白基因的原位定量分析

利用微型计算机控制的荧光显微镜、荧光强度检测仪和图像记录装置并结合荧光原位杂交法对果蝇细胞核内组蛋白基因的复制时期进行了研究,从而建立了一套细胞内直接定量分析的方法。根据果蝇胚胎原代培养细胞核的ＤＡＰＩＩ杂色强度确定处于Ｓ期的细胞。用杂交信号的荧光强度与细胞核荧光强度的相关关系来反映组蛋白基因的复制时期。结果表明果蝇组蛋白基因的复制是在ＤＮＡ合成早期进行的。这套方法至少可直接在细胞上对每套基因组１     

记新一代微型化高分辨率双光子显微镜的诞生——程和平院士专访

正在生物医学领域,成像技术是最重要的技术手段之一,它让研究者能够观察到组织和细胞内正在发生的过程,为各项研究提供直接而确切的证据.传统的单光子荧光显微镜用单个光子将荧光分子激发到激发态,进而产生可被观测的荧光,而发明于1990年的双光子显微镜则是用两个光子来激发同一个荧光分子.与单光子显微镜相比,它所使用的激光波长更长(单个光子的能量更小),具备更高的组织穿透性和更小的光毒性.此外,双光子的激发能力与能     

对果蝇S期细胞内组蛋白基因的原位定量分析

利用微型计算机控制的荧光显微镜、荧光强度检测仪和图像记录装置并结合荧光原位杂交法对果蝇细胞核内组蛋白基因的复制时期进行了研究,从而建立了一套细胞内直接定量分析的方法。根据果蝇胚胎原代培养细胞核的DAPI染色强度确定处于S期的细胞。用杂交信号的荧光强度与细胞核荧光强度的相关关系来反映组蛋白基因的复制时期。结果表明果蝇组蛋白基因的复制是在DNA合成早期进行的。这套方法至少可直接在细胞上对每套基因组100以上拷贝数的熏复DNA序列进行有效的定量分析。     

天然抗氧化剂丹参酮Ⅱ—A对肝细胞脂质过氧化产物与DNA相互作用的影响

［＾３Ｈ］花生四烯酸标记的肝细胞,经ＦｅＣｌ２－ＤＴＰＡ启动脂质过氧化后,细胞ＤＮＡ出现放射性,并随保温时间增加而逐渐增高,表明在细胞内脂质过氧化产物与ＤＮＡ发生相互作用,生成了一种ＤＮＡ加成物,经测定它具有特征荧光光谱,显示较低的增色效应和Ｔｍ值。用高度敏度荧光图象显微镜直接观察发现丹参酮Ⅱ－Ａ经细胞摄取后主要滞留在细胞膜与胞浆中。它能有效地抑制细胞脂质过氧化,减少脂质－ＤＮＡ加成物的产生,并阻     

UVB诱导NIH3T3细胞凋亡时的信号转导机制:Ⅱ.神经酰胺所致细胞内Ca~(2+)和pH的变化

利用粘附式细胞仪（ＡＣＡＳ－５７０）结合相应的荧光探针分别测定了外源性神经酰胺诱导ＮＩＨ３Ｔ３细胞凋亡时胞浆游离Ｃａ＾２＋水平和ＵＶＢ照射ＮＩＨ ３Ｔ３细胞所致细胞内ＰＨ的变化以及神经酰胺的生民对这一变化的影响。结果表明,１．神经酰胺能够导致ＮＩＨ ３Ｔ３细胞胞浆游离Ｃａ＾２＋升高既来源于胞外叠为源于胞内钙池,但外钙内流是引起和维持胞内Ｃａ＾２＋处于高水平所必要条件,ＮＩＨ ３Ｔ３细胞也上存在着两     

心肌细胞钙信号研究进展

近年自激光共聚焦显微镜使用以来,结合膜片钳技术及分子生物学方法,在心肌细胞内的钙信号种类以及在心脏兴奋－收缩偶联研究方面取得了突破性进展。本文介绍了心肌细胞的钙信号研究进展,包括在心肌细胞内可以观察到的钙闪烁,钙微粒,钙波以及由心肌细胞膜上电除极而诱发的瞬时性钙增高等几种心脏细胞内钙变化的形式,意义以及局部调控兴奋－收给偶联的机制。     

Multidimensional stopped-flow photometry monitoring initial processes of platelet activation

A group of initial processes in platelet activation, consisting of a platelet shape change, an intracellular calcium mobilization, a calcium efflux, and a membrane fluidity (mobility) change, has been examined in rabbit platelets by a multidimensional stopped-flow method with light scattering, light transmission, and fluorescence measurements. It was found that a 90 degrees light scattering change and internal calcium release (monitored in terms of chlortetracycline fluorescence) take place after a short lag (5 s at 25 degrees C and 2 s at 37 degrees C) following activation by thrombin. The duration of the lag was the same in both cases. During the initial lag period, a rapid increase in platelet membrane fluidity (mobility) was observed by the use of pyrene excimer fluorescence. These results suggest that the intracellular calcium mobilization and the shape change are triggered by the same rate-determining step, and increase in membrane mobility may play some role in the initial stage of platelet activation before intracellular calcium mobilization occurs.     

A novel role for light in the activation of ribulosebisphosphate carboxylase/oxygenase

The effect of modifying calcium concentration on the expression of the photosynthesis circadian rhythm was examined in Euglena gracilis, Klebs strain Z. Expression of the oxygen evolution rhythm required the presence of both extracellular and intracellular calcium. Several treatments were found to uncouple the rate of the light reactions from the biological clock. In the presence of these chemical agents, the rate of oxygen evolution increased steadily throughout the light portion of the light/dark cycle, instead of showing a peak of activity in the middle of the light cycle. Oxygen evolution was uncoupled from the biological clock when extracellular calcium concentrations were altered by the presence of EGTA or LaCl₃. Uncoupling was also observed when intracellular calcium concentrations were disrupted by the use of Ca²⁺ channel blockers, the intracellular Ca²⁺ antagonist 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, or by disrupting expression of the inositol trisphosphate system. Uncoupling was also observed when the diacylglycerol signaling system, which activates kinase C, was inhibited by acridine orange. The inhibition was reversed by the presence of phorbol esters which activate the kinase. It was concluded that both the inositol trisphosphate and diacylglycerol signaling systems were required for the expression of the oxygen evolution rhythm generated by the biological clock.     

Role of Calcium in Phytochrome-Controlled Nyctinastic Movements of Albizzia lophantha Leaflets

The involvement of Ca(2+) on phytochrome-controlled nyctinastic closure in Albizzia lophantha has been studied by testing the effect of the calcium ionophore 6S-[6alpha(2S(*),3S(*)),8beta(R(*)),9beta,11alpha]-5- methyl-amino)-2-[[3,9,11-trimethyl-8-[-1-methyl-2-oxo-2-(1H-pyrrol-2-yl) ethyl]-1,7-dioxaspiro[5.5]-undec-2yl] methyl]-4-benzoxazolecarboxylic acid (A23187) and the intracellular calcium antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). An external supply of Ca(2+) or calcium ionophore A23187 to the Albizzia leaflets emulates the effect of red light irradiation and counteracts the inhibitory effect of far red light. The intracellular calcium antagonist TMB-8 supplied to Albizzia leaflets inhibits the effect of red light, but had no effect on far red irradiated plants. This suggests a dependence between phytochrome action and intracellular free Ca(2+). We suggest that calcium acts as a phytochrome messenger on control of ion fluxes that drive turgor changes in pulvinular motor cells.     

Intracellular calcium dynamics during photolysis.

The objective of this investigation was to gain a deeper understanding of the intracellular events that precede photolysis of cells. A model system, consisting of malignant melanoma cells pretreated with the calcium sensitive fluorescent dye, Fluo-3, was used to examine the intracellular calcium dynamics in single-cell photolysis experiments. Exposure of the cells to 632 nm laser light in the presence of photosensitizer, tin chlorin e6, resulted in a rise in intracellular calcium. The increase in intracellular calcium was blocked using a variety of calcium channel blocking agents, including verapamil, nifedipine, and nickel. Treatment with the channel blockers was also effective in either decreasing or eliminating cell death despite the presence of lethal doses of photosensitizer and irradiation. These results show that intracellular calcium rises prior to plasma membrane lysis, and that this early rise in intracellular calcium is necessary for membrane rupture.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Calcium mediates the light-induced decrease in maintained K+ current in Limulus ventral photoreceptors

In addition to increasing the conductance to sodium, light reduces the maintained voltage-dependent potassium current (iK) in Limulus ventral photoreceptors. We have investigated the mechanism underlying this long-lasting decrease in ik. Intracellular injection of calcium produced a similar reduction of the voltage-dependent outward current. This reduction was not due to an activation of the voltage-dependent inward current (iin) because calcium injection reduced the outward current even under conditions where iin was blocked with Ni2+, and because calcium injection produced a decrease in conductance, as measured from the slope of the instantaneous i-V curve. The effect of light on ik could be blocked by injection of the calcium buffer EGTA (pCa 7.1) to an intracellular concentration of 50-70 mM. Even larger injections of the pH buffer MOPS (100-200 mM) did not reduce the effect of light on ik. These experiments show that intracellular free calcium (Cai2+) can reduce ik. Furthermore, since Cai2+ is known to increase in light, our results are consistent with the hypothesis that calcium is the internal transmitter for the light-induced decrease in ik.     

He—Ne激光照射对成纤维细胞内[Ca^2＋]i浓度影响的流式细胞计测定

目的：Ｈｅ－Ｎｅ激光照射治疗的机理不明,激光照射引起细胞内Ｃａ＾２＋水平变化,为治疗机理提供理论依据。方法：Ｈｅ－Ｎｅ激光照射引起鼠成纤维细胞Ｌ９２９内［Ｃａ＾２＋］ｉ的变化,用ＨＯ３４２对细胞ＤＮＡ活性染色,Ｆｌｕｏ－３ＡＭ对细胞内Ｃａ＾２＋染色,利用ＦＣＭ同时定量分析细胞ＤＮＡ和细胞内Ｃａ＾２＋的变化。结果：激光照射１５ｍｉｎ（光剂量１１．８１Ｊ／ｃｍ＾２后,ＦＣＭ分析可见ＤＮＡ分布直方图右移     

Regulation of 22Na+ transport by calcium in an established kidney epithelial cell line.

The role of calcium in regulating the Na+ channel in an established kidney epithelial cell line has been examined. Extracellular calcium was inhibitory to Na+ uptake, and a Dixon plot of the initial Na+ uptake rate in the presence of Ca2+ was nonlinear, suggesting a mixed pattern of inhibition. Similar patterns of inhibition were also observed for other divalent cations, including Ba2+, Mg2+, and Mn2+. In contrast elevated concentrations of intracellular calcium resulted in a stimulation of Na+ entry. This intracellular effect was specific to calcium, with Mg2+ and Mn2+ appearing much less effective. Lineweaver-Burk plots of Na+ influx in calcium-loaded and unloaded cells were linear, suggesting that under both conditions a single system transported Na+. Although Na+ entry was stimulated by intracellular Ca2+, the cells did not exhibit other counter transport phenomena reported with cell types in which a Na+/Ca2+ exchange system is operative. Thus, the results indicate that calcium acts as an allosteric regulator of Na+ transport by the Na+ channel.     

二性霉素B与β-escin混合穿孔电极液在人心房肌细胞SK2电流记录中的应用

目的:为研究钙离子对人心房肌细胞小电导钙激活钾通道电流(ISK2)的调节作用,建立用二性霉素B(amphotericin B)与β-escin穿孔的膜片钳(PPR)技术。方法:体外循环术中取得右心耳,应用急性酶分离获得单个人体心房肌细胞,用二性霉素B和/或β-escin作为穿孔电极液,进行穿孔膜片钳实验,在此模式下测试钙离子对人心房肌细胞SK2电流的调控作用,并用胞内钙测试系统验证穿孔前后胞内钙变化。结果:混合使用穿孔电极液6.88μg/mlβ-escin和150μg/ml二性霉素B与单独用150μg/ml二性霉素B或6.88μg/mlβ-escin相比,前一种方法细胞容易封接,能形成稳定的穿孔膜片钳记录模式,可观察到SK2电流有激活,且胞内钙测试系统检测时可观察到穿孔后电极液至细胞内的游离钙离子浓度的增加,F340/380增强。结论:适当浓度的二性霉素B与β-escin混合使用进行穿孔膜片钳实验是一种稳定的全细胞膜片的记录技术,可以用于胞内游离钙离子对SK2电流调控的研究。     

Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors

Single pressure injections of 1-2 mM calcium aspartate into the light-sensitive region of Limulus ventral photoreceptors resulted in a rapid, 20-40-mV depolarization lasting approximately 2 s. The depolarization closely followed the rise in intracellular free calcium caused by the injection, as indicated by aequorin luminescence. The depolarization was followed by reversible desensitization (adaptation) of responses to both light and inositol 1,4,5 trisphosphate. Similar single injections of calcium into the light-insensitive region of the receptor were essentially without effect, even though aequorin luminescence indicated a large, rapid rise in intracellular free calcium. The depolarization caused by injection of calcium arose from the activation of an inward current with rectification characteristics and a reversal potential between +10 and +20 mV that were similar to those of the light-activated conductance, which suggests that the same channels were activated by light and by calcium. The reversal potentials of the light- and calcium-activated currents shifted similarly when three-fourths of the extracellular sodium was replaced by sucrose, but were not affected by a similar replacement of sodium by lithium. The current activated by calcium was abolished by prior injection of a calcium buffer solution containing EGTA. The responses of the same cells to brief light flashes were slowed and diminished in amplitude, but were not abolished after the injection of calcium buffer. Light adaptation and prior injection of calcium diminished the calcium-activated current much less than they diminished the light-activated current.