清热解毒药对血管平滑肌细胞增殖、细胞周期的影响

目的 :观察清热解毒药双花、公英、虎杖、连翘对血管平滑肌细胞生长、贴壁和增殖抑制作用 ,并从对细胞周期的影响角度分析它们的作用机制。方法 :采用细胞计数、MTT法和流式细胞技术。结果 :双花、公英、虎杖、连翘对h PDGF B/B刺激下的平滑肌细胞生长和贴壁均有抑制作用。对平滑肌细胞增殖抑制率分别达 9.2 6%～ 77.35% ,7.78%～ 62 .65% ,5.88%～ 47.81 % ,3.98%～ 33.35% ,其增殖抑制作用呈量效时效关系。通过对细胞周期分析知道 ,双花、公英、虎杖均可以减少 S期 ,增加 G0 /G1期细胞数目。结论 :清热解毒药双花、公英、虎杖、连翘对平滑肌细胞生长及增殖有抑制作用 ,其作用机制可能是通过抑制 DNA合成 ,减少进入 S期细胞数目 ,使细胞增殖停留在 G0 /G1期来实现的     

Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas.

Exocrine gland cells secrete Cl(-)-rich fluid when stimulated by neurotransmitters or hormones. This is generally ascribed to a rise in cytosolic Ca2+ concentration ([Ca2+]i), which leads to activation of Ca2(+)-dependent ion channels. A precise understanding of Cl- secretion from these cells has been hampered by a lack of knowledge about the spatial distribution of the Ca2+ signal and of the Ca2(+)-dependent ion channels in the secreting epithelial cells. We have now used the whole-cell patch-clamp method and digital imaging of [Ca2+]i to examine the response of rat pancreatic acinar cells to acetylcholine. We found a polarization of [Ca2+]i elevation and ion channel activation, and suggest that this comprises a novel 'push-pull' mechanism for unidirectional Cl- secretion. This mechanism would represent a role for cytosolic Ca2+ gradients in cellular function. The cytosolic [Ca2+]i gradients and oscillations of many other cells could have similar roles.     

Phytohaemagglutinin activation of T cells through the sheep red blood cell receptor

Expression of receptors for sheep red blood cells and the ability to proliferate in response to phytohaemagglutinin (PHA) are the traditional properties of human T cells, but the function of the sheep red cell receptor (the T11 antigen) is controversial and the mechanism of PHA-induced mitogenesis unclear. Mitogenesis involves a complex series of cell-mediated and factor-dependent interactions, but a rise in intracellular free calcium concentration, [Ca2+]i, seems to be an important primary event in T-cell activation. We have now investigated the effects of three monoclonal antibodies, previously shown to inhibit mitogen-induced proliferation, on T-cell [Ca2+]i. We find that anti-LFA-2 and OKT11, which react with the sheep red cell receptor, have no effect on [Ca2+]i, nor do they inhibit the rise in [Ca2+]i induced by concanavalin A (Con A) or the mitogenic anti-T3 monoclonal antibody UCHT1 (ref. 11). They do, however, block PHA-induced Ca2+ mobilization. Anti-LFA-1, which reacts with the lymphocyte function-associated antigen, has no effect on intracellular Ca2+. These studies suggest that the sheep red blood cell receptor is an activation pathway for T cells and that the effects of PHA are mediated through this pathway.     

Turning of nerve growth cones induced by localized increases in intracellular calcium ions

Guidance of developing axons involves turning of the motile tip, the growth cone, in response to a variety of extracellular cues. Little is known about the intracellular mechanism by which the directional signal is transduced. Ca2+ is a key second messenger in growth cone extension and has been implicated in growth-cone turning. Here I report that a direct, spatially restricted elevation of intracellular Ca2+ concentration ([Ca2+]i) on one side of the growth cone by focal laser-induced photolysis (FLIP) of caged Ca2+ consistently induced turning of the growth cone to the side with elevated [Ca2+]i (attraction). Furthermore, when the resting [Ca2+]i at the growth cone was decreased by the removal of extracellular Ca2+, the same focal elevation of [Ca2+]i by FLIP induced repulsion. These results provide direct evidence that a localized Ca2+ signal in the growth cone can provide the intracellular directional cue for extension and is sufficient to initiate both attraction and repulsion. By integrating local and global Ca2+ signals, a growth cone could thus generate different turning responses under different environmental conditions during guidance.     

Antigen-dependent increase in cytosolic free calcium in specific human T-lymphocyte clones

Calcium has been implicated as an intracellular messenger in the cellular response to various external stimuli. Exposure of lymphocytes to various mitogens and lectins results in rapid transmembrane calcium fluxes and increased cytoplasmic calcium concentrations ([Ca2+]i). It is not clear, however, whether the mechanisms by which these non-physiological stimuli activate cells are related to those involved in antigen-specific activation. We have now used antigen-specific T-cell clones to study changes in [Ca2+]i associated with specific activation and show here that these cells respond specifically in the presence of antigen and antigen-presenting cells (APC) with increased [Ca2+]i and that this increased [Ca2+]i shows the same genetic restrictions as are seen in the proliferation assay. The kinetics of the [Ca2+]i response to antigen indicate that antigen undergoes a time-dependent processing step as a prerequisite for recognition by T cells, as has been shown for T-cell proliferative responses, but that the [Ca2+]i response to processed antigen is extremely rapid. The close correlation between changes in [Ca2+]i and cell activation resulting in proliferation suggests that Ca2+ may act as an intracellular messenger in antigen-specific responses.     

Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes

Compelling evidence has existed for more than a decade for a sodium/calcium (Na-Ca) exchange mechanism in the surface membrane of mammalian heart muscle cells which exchanges about three sodium ions for each calcium ion. Although it is known that cardiac muscle contraction is regulated by a transient increase in intracellular calcium ([Ca2+]i) triggered by the action potential, the contribution of the Na-Ca exchanger to the [Ca2+]i transient and to calcium extrusion during rest is unclear. To clarify these questions, changes in [Ca2+]i were measured with indo-1 in single cardiac myocytes which were voltage clamped and dialysed with a physiological level of sodium. We find that Ca entry through the Na-Ca exchanger is too slow to affect markedly the rate of rise of the normal [Ca2+]i transient. On repolarization, Ca extrusion by the exchanger causes [Ca2+]i to decline with a time constant of 0.5 s at -80 mV. The rate of decline can be slowed e-fold with a 77-mV depolarization. Calcium extrusion by the exchanger can account for about 15% of the rate of decline of the [Ca2+]i transient (the remainder being calcium resequestration by the sarcoplasmic reticulum (SR]. The ability of the cell to extrude calcium was greatly reduced on inhibiting the exchanger by removing external sodium, which itself led to an increase in resting [Ca2+]i. This finding is in contrast to the suggestion that calcium extrusion at rest is mediated mainly by a sarcolemmal Ca-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

加热对血管平滑肌细胞的影响实验研究

 为支架磁感应热疗预防和治疗PTCA术后血管再狭窄提供基础研究数据,对临床常用的316L型不锈钢冠脉支架进行磁感应诱导升温,探讨加热对兔血管平滑肌细胞（VSMCs）的增殖、迁移、凋亡及周期的影响。将平滑肌细胞置于恒温水浴槽中,待其达到预定温度（43、47℃）后持续10min,观察细胞形态变化,采用MTT法检测加热对血管平滑肌细胞增殖活性的影响,流式细胞术检测VSMC细胞周期和细胞凋亡,划痕实验检测加热对细胞迁移能力的影响,免疫细胞化学法检测不同温度作用后血管平滑肌细胞中增殖细胞核抗原（PCNA）的表达。结果发现,支架在交变磁场下可以升温至热疗所需温度,加热后细胞增殖受到了显著抑制,43℃组细胞存活率为（83.23±2.87）%,47℃组细胞存活率仅为（37.58±0.78）%。细胞的凋亡率显著增加,47℃组细胞凋亡率为（87.37±2.95）%,与对照组相比具有极显著差异,而43℃组的凋亡率为（6.00±0.26）%,与对照组相比无统计学差异。细胞周期也受到抑制,被阻滞在S期。加热后随着时间的推移,47℃组细胞的迁移能力受到显著抑制,而加热对43℃组细胞的迁移能力无显著影响。免疫细胞化学检测显示,随着温度的升高,PCNA的表达受到明显抑制。研究表明,临床常用冠脉支架在交变磁场下升温可行。加热显著抑制了细胞的增殖并促进了细胞凋亡,使细胞周期阻滞在S期,且抑制了细胞的迁移。加热能显著抑制细胞PCNA的表达,这些可能是加热抑制细胞增殖和迁移、促进凋亡的机制之一。     

QKI对血管平滑肌细胞增殖的抑制功能

观察QKI蛋白对血管平滑肌细胞(VSMCs)增殖的影响,进一步完善血管平滑肌细胞增殖的相关机制.以大鼠的原代主动脉平滑肌细胞为研究对象,观察分别过表达QKI5、QKI6对血清促增殖作用的影响.细胞增殖采用MTT法和Western blot测定.结果血清可促进大鼠的原代主动脉平滑肌细胞增殖,过表达QKI可抑制血清刺激的血管平滑肌细胞的增殖.说明过表达QKI对血清刺激的血管平滑肌细胞增殖具有抑制作用,该作用有可能在一定程度上能够抑制高血压等病伴发的血管平滑肌增殖.     

Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca^2＋ transport proteins and the effect of taurine on myocardial protection in rabbits

To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca^2 transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes ( Myo [ Ca^2 ]i ), activity of SR Ca^2 -ATPase (SERCA2a) , level of SERCA2a mRNA and Ca^2 released channels(RYR2) mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca^2 -ATPase and level of SERCA2a mRNA decreased , while Myo[ Ca^2 ]i increased in DOX-treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[ Ca^2 ] i and the decrease of SERCA2a mRNA induced by doxorubicin. Tile results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX-induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.     

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.     

核糖体蛋白RPL34-PS1对B细胞淋巴瘤的生长促进作用

一种核糖体蛋白RPL34具有促进B细胞淋巴瘤生长的作用,通过分子克隆将RPL34基因的序列克隆到逆转录病毒载体pBABE-Puro上后,包装逆转录病毒并感染Balb/c小鼠来源的B淋巴瘤细胞38B9,经嘌呤霉素筛选后构建稳定过表达RPL34的B淋巴瘤细胞系RPL34-38B9。体外培养计数比较细胞增殖变化情况;流式检测细胞凋亡和细胞周期水平;小鼠皮下荷瘤并测量肿瘤体积。结果表明:过表达的核糖体蛋白RPL34能够显著促进B淋巴瘤细胞的生长速率,减少细胞凋亡,同时能够显著增加B淋巴瘤细胞的成瘤速率。     

啤酒花中蛇麻酮诱导细胞凋亡及机制研究

通过体外抗肿瘤实验观察啤酒花(Humulus LupulusL.)中成分蛇麻酮(lupulone)对肿瘤细胞SGC-7901和HepG-2诱导细胞凋亡的作用,并揭示其作用机制.采用MTT法观察蛇麻酮体外抗肿瘤作用;采用流式细胞仪观察蛇麻酮对肿瘤细胞周期及细胞凋亡的影响;采用Fluo-3AM探针标记,激光共聚焦技术观测细胞内[Ca2 ]i变化.蛇麻酮对肿瘤细胞SGC-7901及HepG-2均有较好的疗效.蛇麻酮作用于SGC-7901和HepG-2细胞48 h后,肿瘤细胞均有明显凋亡峰出现.蛇麻酮可以将肿瘤细胞SGC-7901和HepG-2的细胞周期阻滞在G0/G1和S细胞期,升高细胞凋亡指数(APO).蛇麻酮可使SGC-7901细胞内[Ca2 ]i升高,使HepG-2细胞内[Ca2 ]i先升高后降低.蛇麻酮通过诱导细胞凋亡来发挥抗肿瘤作用.蛇麻酮通过升高肿瘤细胞内[Ca2 ]i启动肿瘤细胞凋亡机制,[Ca2 ]i升高时Ca2 来源于细胞内钙库释放.     

Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells.

Rat basophilic leukaemia cells, like mast cells from which they are derived, have surface Fc epsilon receptors that trigger secretion of inflammatory mediators when crosslinked. Both GTP-binding proteins and a rise in cytosolic calcium concentration ([Ca2+]i) are implicated in the secretory mechanism. Here we use a video-imaging technique to report that transient rises in [Ca2+]i initiated in an individual cell can spread from cell to cell in a wave-like pattern by means of a secreted intermediate, in the absence of gap-junctional communication. We find that the leukaemia cells, peritoneal mast cells and mucosal mast cells have cell-surface P2-type purinergic receptors that can trigger similar [Ca2+]i transients. We provide evidence that ATP is rapidly released, and that it can amplify [Ca2+]i signals and initial secretory responses during antigen-stimulation of rat basophilic leukaemia cells.     

Effects of ATP and vanadate on calcium efflux from barnacle muscle fibres

Calcium ions carry the inward current during depolarization of barnacle muscle fibres and are involved in the contraction process. Intracellular ionized calcium ([Ca2+]i) in barnacle muscle, as in other cells, is kept at a very low concentration, against a large electrochemical gradient. This large gradient is maintained by Ca2+ extrusion mechanisms. When [Ca2+]i is below the contraction threshold, Ca2+ efflux from giant barnacle muscle fibres is, largely, both ATP dependent and external Na+ (Na+0) dependent (see also refs 5,6). When [Ca2+]i is raised to the level expected during muscle contraction (2-5 muM), most of the Ca2+ efflux from perfused fibres is Na0 dependent; as in squid axons, this Na+0-dependent Ca2+ efflux is ATP independent. Orthovanadate is an inhibitor of (Na+ + K+) ATPase and the red cell Ca2+-ATpase. We report here that vanadate inhibits ATP-promoted, Na+0-dependent Ca2+ efflux from barnacle muscle fibres perfused with low [Ca2+]i (0.2-0.5 microM), but has little effect on the Na+0-dependent, ATP-independent Ca2+ efflux from fibres with a high [Ca]i (2-5 microM). Nevertheless, ATP depletion or vanadate treatment of high [Ca2+]i fibres causes an approximately 50-fold increase of Ca2+ efflux into Ca2+-containing lithium seawater. These results demonstrate that both vanadate and ATP affect Ca2+ extrusion, including the Na+0-dependent Ca2+ efflux (Na-Ca exchange), in barnacle muscle.     

Repetitive spikes in cytoplasmic calcium evoked by histamine in human endothelial cells

Measurement of cytoplasmic free calcium, [Ca2+]i, in single human endothelial cells has shown that low doses of the inflammatory mediator histamine evoke asynchronous repetitive spikes in [Ca2+]i whereas high doses cause a maintained elevated [Ca2+]i. We discuss possible regulatory mechanisms, and the potential physiological and pathological implications of such a frequency-modulated [Ca2+]i signalling system.     

依地福新抑制Jurkat细胞生长机制的研究

目的观察依地福新对体外培养的Jurkat细胞的生长抑制作用,并进一步探讨其作用机制。方法应用MTT法检测细胞增殖活性;流式细胞仪检测细胞周期分布;Annexin V—FITC染色法检测细胞凋亡率。结果不同浓度依地福新处理Jurkat细胞24～96h后,细胞增殖显著受到抑制,并呈现浓度及时间依赖性;1．0μmol／L、5．0ymol／L、10．0μmol／L依地福新处理72h后,Jurkat细胞G0／G1期细胞数量显著增加,S期细胞数量显著降低（P〈0．01）;各浓度组细胞凋亡率均显著增加（P〈0．01）。结论依地福新对Jurkat细胞具有生长抑制作用,其机制与阻滞细胞周期及诱导凋亡有关。     

Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion 'willingness' in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (approximately 4) is gradually reduced below 3 microM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.     

Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels

It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.     

Platelet activation--a role for a 40K anti-phospholipase A2 protein indistinguishable from lipocortin

Stimulus-response (S-R) coupling in platelets requires an intermediary other than an elevation in cytosolic free calcium ([Ca2+]i). While an increase in [Ca2+]i is essential in S-R coupling, effecting phosphorylation of myosin of relative molecular mass (Mr) 20,000 (20 K), platelet activation is also associated with phosphorylation of a 40K protein, which can occur in the absence of changes in [Ca2+]i. The 40K protein is the substrate for protein kinase C (PKC). Mounting evidence suggests that activation of PKC by diacylglycerol is the other signal involved in S-R coupling. Although phosphorylation of the 40K protein is associated with certain platelet functional responses, no precise role has been accredited to it. Recently, we and others have described several proteins (collectively known as lipocortin) which inhibit phospholipase A2 (PLA2). One of the most conspicuous proteins of this group is a 40K peptide whose inhibitory activity can be suppressed by prior phosphorylation. We hypothesized that the 40K protein described in platelets may possess anti-PLA2 activity and that phosphorylation by PKC, suppressing its inhibitory activity, may represent the mechanism underlying mobilization of arachidonic acid, the precursor of prostaglandins. The results of the present study strongly support this hypothesis.     

Diacylglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets

An increase in cytoplasmic free calcium, [Ca2+]i, is thought to be the trigger for secretory exocytosis in many cells. In blood platelets, large rises in [Ca2+]i can cause secretion and calcium has been regarded as the final common activator not only for secretion but also for shape-change and aggregation. We have shown that while thrombin and platelet-activating factor (PAF) normally elevate [Ca2+]i, they can also stimulate shape-change and secretion even when the [Ca2+]i rise is suppressed. The present results strongly implicate diacylglycerol, produced by stimulus-dependent breakdown of phosphoinositide, in this calcium-independent activation. Exogenous diacylglycerol activates a protein kinase (C-kinase) in platelets as do PAF, thrombin and collagen. 12-O-tetradecanoyl phorbol-13-acetate (TPA) also activates C-kinase and is a potent stimulus for secretion and aggregation. It is shown here that the exogenous diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG) and TPA evoke similar secretion and aggregation without elevating [Ca2+]i above the basal level of 0.1 microM. The pattern of secretion resembles that produced by collagen and thrombin when [Ca2+]i remains at basal levels. Modest increases in [Ca2+]i, insufficient to stimulate secretion, markedly accelerate the responses to TPA and OAG.