槲皮素磷酸钾对大鼠脑血栓形成模型血小板聚集及血浆TXB_2和6-keto-PGF_(12)水平的影响

以大鼠脑血栓形成为模型,观察槲皮素磷酯钾对大鼠脑血栓形成术24h后血小板聚集、血浆TxB2和6-keto-PGF12水平的影响,以及脑电图、脑重量和病理组织学改变。结果表明:复合血栓诱导剂1ml kg-1经颈内动脉注射能诱发大鼠同侧大脑半球内血栓形成,槲皮素磷酯酶钾10-20 mg kg-1能对抗大鼠脑血栓形成,降低体内血小板自发性聚集,抑制血浆TxB2的升高。提示槲皮素磷酸酯钾可能通过其抗血小板聚集及影响花生回烯酸代谢而发挥抗脑血栓形成作用。     

内毒素所致DIC时血小板胞浆Ca^2＋浓度变化的研究

本实验观察内毒素在体外对兔血小板胞浆游离钙浓度[Ca~(2+)]i的作用以及用二次注射内毒素法复制DIC模型时对[Ca~(2+)]i的影响。结果表明静息血小板[Ca~(2+)]i为112±24 nM,内毒素可直接作用于血小板,使[Ca~(2+)]i呈剂量依赖性升高。内毒素致DIG时(Ca~(2+)]i可升高三倍。结果提示内毒素可能是通过升高血小板[Ca~(2+)]i而激活血小板。血小板的激活可能是导致DIC的重要发病机制之一。     

当归与硝苯吡啶对慢性支气管炎肺泡巨噬细胞胞浆游离钙水平的影响

目的:探讨当归与硝苯吡啶对慢性支气管炎(慢支)肺泡巨噬细胞胞浆游离钙水平的影响。方法:对慢支缓解期患者7例和正常对照者6例进行支气管肺泡灌洗获得的肺泡巨噬细胞经分离、纯化后,加Fura-2/AM负载,以Fura-2荧光比值法测定加当归、硝苯吡啶及LPS后胞浆游离钙的水平。结果:慢支组肺泡巨噬细胞胞浆中基础钙水平(189.47±23.69)nmol/L较正常对照组(99.65±32.21)nmol/L明显增高(P＜0.01);LPS促进慢支组肺泡巨噬细胞包括胞内钙库释放引起的胞浆游离钙水平升高(基础钙189.47±23.69nmol/L;LPS组235.53±30.30nmol/L)(静息钙228.41±27.36nmol/L;氯化钙+LPS组288.47±43.68nmol/L)(P均＜0.01);当归及硝苯吡啶均抑制LPS对慢支组肺泡巨噬细胞胞浆游离钙水平的升高作用(静息钙228.41±27.36nmol/L;氯化钙+当归+LPS组236.68±28.60nmol/L;氯化钙+硝苯吡啶+LPS组252.64±37.05nmol/L)(P均＞0.05)。结论:当归与硝苯吡啶通过抑制慢支患者肺泡巨噬细胞胞浆游离钙水平升高,影响肺泡巨噬细胞的活化,对于慢支气道内的非特异性炎症可能具有抑制作用。     

淋巴细胞经TCR—CD3活化增殖作用的分析

本文探讨了抗ＣＤ３单抗诱导的淋巴细胞活化增殖及有关影响因素。实验结果表明：①淋巴细胞内钙升高是淋巴细胞活化增殖的重要条件，ＣＤ３ＭｃＡｂ引起的早期胞浆游离钙迅速升高主要由内质网释放钙离子所致，而淋巴细胞增殖不仅需要细胞内钙释放，还需要细胞外钙内流；②ＧＴＰ结合蛋白是淋巴细胞激活过程的一重要环节，经Ｇ蛋白作用物霍乱毒素作用后，淋巴细胞ＤＮＡ合成显著降低；③新霉素和ＰＳＳ可抑制ＰＬＣ和ＰＫＣ的活性，对     

心肌细胞收缩期钙瞬变的调节机制的研究进展

心肌细胞收缩是由细胞内游离钙浓度([Ca^2 ]i)收缩期增高引起的。[Ca^2 ]i增高的多少是心肌收缩力量和心输出量的主要调节机制。而胞浆收缩Ca^2 主要来自于胞外钙内流和肌浆网(sacroplasmic reticulum，SR)的钙释放。本介绍心肌细胞收缩胞浆Ca^2 浓度升高的机制与部分调节因素的分子研究进展。     

脂氧素A4通过抑制Jurkat T细胞钙池操纵的钙通道电流而抑制其激活和增殖

目的通过研究脂氧素A4（LXA4）对Jurkat T细胞内游离钙离子浓度变化、钙池操纵的钙通道电流（Isoc）及细胞激活和增殖的影响，初步探讨其对T细胞的调节作用及机制。方法钙离子荧光探针Fluo-3／AM负载细胞后，用激光共聚焦扫描显微镜技术动态检测活细胞内游离钙离子浓度的变化；全细胞膜片钳技术记录Isoc；ELISA测IL-2水平；3^H-TdR掺入法测细胞增殖情况，并结合流式细胞仪进行细胞周期分析。结果（1）用含钙细胞外液时，LXA4降低正常Jurkat T细胞内游离钙离子浓度，而用无钙细胞外液时则无显著差异；（2）LXA4呈剂量依赖性抑制钙池操纵的钙通道（SOC）激活剂thapsigargin（TG）引起Jurkat T细胞内游离钙浓度的增高，100nmol／L LXA4也能抑制anti-CD3或植物血凝素（PHA）引起的细胞内钙离子浓度的增高，SOC阻滞剂2-aminoethoxydiphenylborate（2-APB）能显著抑制TG引起的钙内流；（3）膜片钳结果显示，LXA4抑制正常Jurkat T细胞Isoc，TG能显著增大Isoc，而LXA4呈剂量依赖性抑制TG引起的Isoc的升高；（4）LXA4剂量依赖性抑制anti-CD3和anti-CD28诱导的Jurkat T细胞IL-2表达及细胞增殖，细胞周期分析发现LXA4阻止Jurkat T细胞由GI1期进入S期，降低S期细胞比例。结论LXA4可能通过抑制Jurkat T细胞Isoc引起的胞质内游离钙离子浓度的升高而抑制其激活和增殖。     

PTCA围手术期硫氮(艹卓)酮对血小板活性和钙动力学的影响

目的 :研究硫氮  酮对内皮损伤后血小板激活的影响及与胞浆Ca2 浓度的关系。方法 :35例拟行PTCA治疗的心绞痛患者随机分成对照 (C)组和硫氮 艹卓 酮治疗 (D)组 ,由冠状静脉窦和外周静脉采血 ,检测围术期血小板GPⅡb/Ⅲa表达和胞浆Ca2 浓度。并在体外研究不同浓度硫氮 艹卓 酮对稳定型心绞痛患者全血GPⅡb/Ⅲa表达的影响。结果 :C组首次球囊扩张后 5min、10min和PTCA后 10min时GPⅡb/Ⅲa表达和 [Ca2 ]i明显升高 ,而D组未见升高。高于治疗浓度的硫氮 艹卓 酮 (2 0 0 μg/L以上 )显著抑制GPⅡb/Ⅲa表达。 结论 :硫氮 艹卓 酮和阿司匹林 /抵克力得联合治疗可防止常规阿司匹林 /抵克力得治疗时发生的血小板激活。硫氮 艹卓 酮抗血小板作用可能是抑制[Ca2 ]i升高的结果 ,但只在高于治疗浓度时有效     

三磷酸肌醇亦可使三磷酸肌醇敏感性钙通道失活

许多细胞外信号通过1,4,5-三磷酸肌醇(IP_3)激活细胞钙贮池膜钙通道使内钙释放和胞浆游离钙浓度[Ca~(2+)]i增加。生理条件下[Ca~(2+)]i不会持续升高,其机制除有细胞膜钙ATP酶作用外,同钙通道失活也有关。曾认为,Ca~(2+)-钙调蛋白复合物及Ca~(2+)可致通道失活,但本实验提示:IP_3本身也     

人参皂甙Rd对皮层神经元兴奋性毒性损伤后胞内游离钙的影响

目的:探讨人参皂甙Rd(Ginsenoside Rd,GSRd)对皮层神经元兴奋性毒性损伤后细胞内游离钙离子浓度变化的影响。方法:采用原代方法培养大鼠皮层神经元,免疫荧光染色鉴定神经元纯度。应用激光共聚焦显微镜,观察GSRd对谷氨酸(Glutamate,Glu)和N-甲基-D-天门冬氨酸(NMDA)刺激后神经元胞内游离钙离子浓度变化的影响。使用钙离子荧光探针Fluo-4,AM标记细胞内游离钙,以Fluo-4的荧光强度反映细胞内游离钙浓度变化。结果:空白对照组荧光强度没有明显变化,而高浓度Glu刺激可迅速升高神经元胞内的荧光强度;在给予GSRd干预时,荧光强度升高的幅度明显降低,与MK-801的作用相似;NMDA刺激亦可使神经元胞内荧光强度明显升高,而加入GSRd干预时,荧光强度升高的幅度较NMDA损伤组有明显减小。结论:GSRd能够抑制高浓度Glu和NMDA引起的大量钙内流,提示减轻兴奋性毒性损伤过程中的钙超载可能是GSRd神经保护作用的机制之一。     

淋巴细胞经TCR－CD3活化增殖作用的分析

本文探讨了抗ＣＤ３单抗诱导的淋巴细胞活化增殖及有关影响因素。实验结果表明：①淋巴细胞内钙升高是淋巴细胞活化增殖的重要条件，ＣＤ３ＭｃＡｂ引起的早期胞浆游离钙迅速升高主要由内质网释放钙离子所致，而淋巴细胞增殖不仅需要细胞内钙释放，还需要细胞外钙内流；②ＧＴＰ结合蛋白是淋巴细胞激活过程的一重要环节，经Ｇ蛋白作用物霍乱毒素作用后，淋巴细胞ＤＮＡ合成显著降低；③新霉素和ＰＳＳ可抑制ＰＬＣ和ＰｋＣ的活性，对淋巴细胞ＮＤＡ合成造成剂量依赖性抑制作用。此外，抗ＣＤ３ＭｃＡｂ诱导的淋巴细胞ＤＮＡ合成需要辅佐细胞的存在，高度纯化的Ｔ细胞对ＣＤ３ＭｃＡｂ的刺激不发生增殖反应。     

Characterization of platelet cytoplasmic Ca2+ mobilization in patients with congenital cyclo-oxygenase deficiency and with defective platelet aggregation to A23187

Agonist-induced platelet cytoplasmic Ca2+ concentrations ([Ca2+]i) in patients with congenital cyclo-oxygenase deficiency (A) and with impaired aggregation to A23187 (B) were measured with aequorin in the presence or absence of extracellular Ca2+. The influence of TMB-8 or ONO3708 on agonist-induced [Ca2+]i in those platelets was also investigated. In Patient 1, there was a single aequorin luminescence peak in response to arachidonate, which was a thromboxane A2(TXA2) independent Ca2+ influx. The luminescence peak due to the formation of TXA2 was not detectable. The A23187-induced [Ca2+] i was decreased in the presence of extracellular Ca2+, but was within normal limits in the absence of extracellular Ca2+. A thrombin or STA2-induced elevation of [Ca2+] i was always within normal limits under any conditions. These results suggest that cyclo-oxygenase activity (CO activity) contributes to the A23187-induced Ca2+ influx, but does not contribute to the Ca2+ release from intracellular stores, and that the thrombin or STA2-induced Ca2+ influx and release do not depend on the CO activity. In Patient 2, the time lag from the addition of A23187 to the aequorin luminescence peak was found both in the presence and absence of extracellular Ca2+, which was more obvious in the latter. This A23187-induced elevation of [Ca2+] i disappeared after treatment of the platelets with TMB-8 in the absence of extracellular Ca2+, which is rarely seen in normal platelets. The most striking finding was that the thrombin-induced rise in [Ca2+] i in the absence of extracellular Ca2+ was not detectable. These findings might be closely related to abnormal platelet function in this patient.     

Low intracellular calcium concentration in the platelets after stimulation in patients with myeloproliferative disorders

Intracellular calcium levels in the platelets of patients with myeloproliferative disorders (MPD) were measured by the Aequorin method. In the patients with MPD, the maximum intracellular calcium level [Ca2+]i was significantly lower and the time required to reach the peak calcium level was significantly longer than in normal controls after thrombin (0.5 U/ml) and collagen (2.5 micrograms/ml) stimulation. The rate of increase in intracellular calcium levels after thrombin (0.5 U/ml) stimulation in the presence of various concentrations of extracellular calcium was lower than in the normal controls, which suggests that the low level of intracellular [Ca2+]i after stimulation is caused mainly by a low Ca2+ influx. These results suggest the relationship between abnormalities of membrane glycoprotein and the impaired calcium influx of MPD platelets.     

地塞米松诱导小鼠腹腔巨噬细胞凋亡过程中[Ca~(2 )]i的变化

目的　研究地塞米松诱导凋亡小鼠腹腔巨噬细胞 [Ca2 ]i的变化及其对凋亡的影响以及信使分子对凋亡和 [Ca2 ]i变化的影响。方法　激光扫描共聚焦显微术、流式细胞术和荧光标记术。结果　 1 凋亡巨噬细胞内fluo 3荧光强度逐渐增强。胞内钙库受体抑制剂 ,尤其是Ca2 内流阻断剂抑制细胞内fluo 3荧光强度变化 ,同时使细胞凋亡率降低 ;2 .staurosporine和DcAMP显著降低巨噬细胞凋亡率并明显抑制fluo 3荧光强度改变。genistein和亚甲蓝稍降低巨噬细胞凋亡率 ,并降低fluo 3荧光强度升高幅度。结论　 1 胞外Ca2 内流和内源性Ca2 释放 ,主要是胞外Ca2 内流使[Ca2 ]i逐渐升高并促进巨噬细胞凋亡 ;2 .PKC促进 [Ca2 ]i升高和巨噬细胞凋亡。cAMP抑制[Ca2 ]i升高和巨噬细胞凋亡。cGMP、TPK降低 [Ca2 ]i升高幅度并稍抑制巨噬细胞凋亡。结果提示 ,[Ca2 ]i是信使分子调控巨噬细胞凋亡的主要靶点。     

Calcium-induced calcium release participates in cell volume regulation of rabbit TALH cells

Changes of cell volume and intracellular calcium concentration ([Ca(2+)](i)) in immortalized thick ascending limb of Henle's loop (TALH) cells were monitored using confocal laser scanning microscopy and fura-2 fluorescence, respectively. Reduction of the extracellular osmolarity from 600 to 300 mosmol/l induced cell swelling followed by regulatory volume decrease (RVD). Simultaneously, the [Ca(2+)](i) increased transiently. The calcium rise was not observed in calcium-free solution or in the presence of nifedipine, indicating that the change was, in the first place, due to the activation of a calcium influx. Application of ATP or caffeine in isotonic solutions increased transiently the [Ca(2+)](i) which revealed the existence of stores in TALH cells sensitive to inositol-1,4,5 trisphosphate (IP(3)) and ryanodine. To examine the possibility that the calcium influx might induce calcium release, manganese quenching experiments were performed. In hypotonic calcium-free solutions, the decay of the calcium-insensitive and calcium-sensitive fluorescence occurred simultaneously. In the presence of extracellular calcium however, the calcium-sensitive wavelength revealed initial calcium influx followed by a calcium release from intracellular stores. Thus, the calcium influx was a prerequisite for the calcium release. We conclude that calcium-induced calcium release participates in global calcium signalling during RVD of TALH cells.     

低氧与缺血诱导培养海马和皮质神经元钙应答反应的比较

目的：钙作为重要信使参与多种生理和病理代谢过程,而且在缺血性神经元损伤机制中起着重要的作用。本实验模拟脑缺血的病理生化改变,用激光扫描共聚焦显微镜（LSCM）和Fluo-3荧光探针标记技术,观察低氧缺血状态下体外培养的海马,皮质神经元内钙浓度的变化。方法：用100μmol/L氰化钠造成细胞低氧;100μmol/L氰化钠和3.5mmol/L碘醋酸盐模拟在体完全性脑缺血;1mmol/LL-谷氨酸模拟在体脑缺血时兴奋性氨基酸大量释放;无葡萄糖介质剥夺细胞能量代谢的底物。结果：低氧使海马神经元[Ca^2 ]i显著升高,但有两种不同的钙振荡现象,谷氨酸引起海马神经元[Ca^2 ]i持续升高,但峰值低于低氧组,缺血组未见[Ca^2 ]i大幅升高,葡萄糖缺如不引起[Ca^2 ]i升高,结论：低氧和谷氨酸引起的神经元损害是能量依赖性的,轻度酸中毒可阻止胞内Ca^ 升高,糖对神经元具有保护作用,但单纯无糖引起的神经元损害与Ca^2 超载机制无关。     

Cytosolic calcium alterations in platelets of patients with early stages of Alzheimer's disease

Alterations in calcium homeostasis might be implicated in the neuropathology of Alzheimer's disease (AD). To date it is not clear whether changes in cytosolic calcium level ([Ca2+ ]i) are the result or the cause of pathogenic effects. In platelets of patients with early stages of AD, the basal values of [Ca2+]i in the absence of extracellular Ca2+ were significantly lower in comparison with age-matched and young controls. After the addition of 1 mM calcium into the incubation medium the [Ca2+]i markedly increased in platelets of AD patients whereas the increase only to a smaller extent was observed in control age-matched and young subjects. The present study proposes that calcium dysregulation during the whole disease period could not be uniform and according to our results the [Ca2+]i is reduced in the first stages of AD. We suggest that the disturbed calcium homeostasis in AD is an "early defect."     

Mobilization of calcium by the brief application of oxytocin and prostaglandin E2 in single cultured human myometrial cells.

Intracellular calcium ([Ca2+]i) mobilization was studied in single cultured human myometrial cells in response to the agonists oxytocin and prostaglandin E2 (PGE2) using the fluorescent dye Fura-2. Oxytocin and PGE2 applications were associated with an increase in [Ca2+]i, although there was a marked intercell variation in the amplitude of the agonist-induced response. Removal of extracellular calcium ([Ca2+]o) reduced the oxytocin-induced rise and abolished the PGE2-induced rise in [Ca2+]i, thereby demonstrating that oxytocin but not PGE2 can mobilize intracellular stores of calcium. In nominally calcium-free medium, [Ca2+]i was not increased by PGE2 but subsequent application of oxytocin increased [Ca2+]i, thereby demonstrating that, within a single cell, calcium stores were mobilized by oxytocin and not PGE2. The intracellular calcium stores were completely depleted by a single application of oxytocin and not replenished in the absence of [Ca2+]o. Perfusion with calcium-containing medium for 100 s enabled store refilling. Cell depolarization by 140 mM-K+ caused a transient increase followed by a sustained elevation of [Ca2+]i on which were superimposed small fluctuations. Oxytocin caused an influx of calcium in cells depolarized by K+. This was more marked than that obtained with PGE2.     

地塞米松对谷氨酸升高海马神经元胞内[Ca~(2+)]i作用的影响

为了研究谷氨酸致痫和人工合成的糖皮质激素地塞米松抑痫作用的细胞内机制,本文在ＥＰＣ ９ 光电联合检测系统上用Ｆｕｒａ ２ 阳离子检测法观察了地塞米松对谷氨酸引起的培养乳鼠海马神经细胞内［Ｃａ２＋ ］ｉ 的影响。结果：（１）谷氨酸引起海马神经元内［Ｃａ２＋ ］ｉ 显著升高,ＥＧＴＡ（５ ｍ ｍ ｏｌ／Ｌ）耗竭细胞外钙后,谷氨酸升钙作用消失,给予氯化钙（１ ｍ ｍ ｏｌ／Ｌ）后其升钙作用恢复：Ｖｅｒａｐａｍ ｉｌ（１０ μｍ ｏｌ／Ｌ）对谷氨酸升钙作用无明显的影响,ＭＫ ８０１（１０ μｍ ｏｌ／Ｌ,ＮＭ ＤＡ 受体特异性非竞争性阻断剂）可明显阻断谷氨酸的升钙作用。（２）地塞米松（１００ μｍ ｏｌ／Ｌ）作用２ ｈ 明显抑制了谷氨酸（２００ μｍ ｏｌ／Ｌ）的升钙作用,地塞米松（１００ μｍ ｏｌ／Ｌ）＋ 放线菌酮（１０ μｍ ｏｌ／Ｌ,蛋白合成抑制剂）共同作用２ ｈ,再加入谷氨酸,则地塞米松的抑制作用消失,地塞米松（１００ μｍ ｏｌ／Ｌ）作用２ ｍ ｉｎ 对谷氨酸（２００ μｍ ｏｌ／Ｌ）的升钙作用无明显影响。本实验结果提示,谷氨酸通过ＮＭ ＤＡ 受体介导的外钙内流升高了海马神经元胞内［Ｃａ２＋ ］ｉ,地塞米松可能通过基因组机制抑制了谷氨酸的这种升     

NMDAR1单克隆抗体抑制谷氨酸诱导的大鼠海马神经元Ca2+内流

目的观察人N-甲基-D-门冬氨酸受体(NMDAR,NR)主亚基(NR1)单克隆抗体mAbN1对谷氨酸诱导的大鼠海马神经元Ca2 内流的影响。方法建立谷氨酸介导的大鼠海马神经元兴奋毒性损伤模型,以mAbN1及MK-801分别预处理海马神经元,用Fluo-3/AM法,在激光扫描共聚焦显微镜下观察对细胞内游离Ca2 浓度([Ca2 ]i)的影响。结果mAbN1能显著抑制谷氨酸所致海马神经元[Ca2 ]i升高,此作用强于MK-801,且其本身对生理状态下神经元[Ca2 ]i无影响。结论mAbN1的抗兴奋毒性作用可能是通过改变NR的蛋白质二级结构从而影响兴奋毒性作用中的Ca2 内流实现的。     

Characterization of the mGluR(1)-mediated electrical and calcium signaling in Purkinje cells of mouse cerebellar slices

The metabotropic glutamate receptor 1 (mGluR(1)) plays a fundamental role in postnatal development and plasticity of ionotropic glutamate receptor-mediated synaptic excitation of cerebellar Purkinje cells. Synaptic activation of mGluR(1) by brief tetanic stimulation of parallel fibers evokes a slow excitatory postsynaptic current and an elevation of intracellular calcium concentration ([Ca2+](i)) in Purkinje cells. The mechanism underlying these responses has not been identified yet. Here we investigated the responses to synaptic and direct activation of mGluR(1) using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca2+](i) in mouse Purkinje cells. Following pharmacological block of ionotropic glutamate receptors, two to six stimuli applied to parallel fibers at 100 Hz evoked a slow inward current that was associated with an elevation of [Ca2+](i). Both the inward current and the rise in [Ca2+](i) increased in size with increasing number of pulses albeit with no clear difference between the minimal number of pulses required to evoke these responses. Application of the mGluR(1) agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) by means of short-lasting (5-100 ms) pressure pulses delivered through an agonist-containing pipette positioned over the Purkinje cell dendrite, evoked responses resembling the synaptically induced inward current and elevation of [Ca2+](i). No increase in [Ca2+](i) was observed with inward currents of comparable amplitudes induced by the ionotropic glutamate receptor agonist AMPA. The 3,5-DHPG-induced inward current but not the associated increase in [Ca2+](i) was depressed when extracellular Na+ was replaced by choline, but, surprisingly, both responses were also depressed when bathing the tissue in a low calcium (0.125 mM) or calcium-free/EGTA solution. Thapsigargin (10 microM) and cyclopiazonic acid (30 microM), inhibitors of sarco-endoplasmic reticulum Ca2+-ATPase, had little effect on either the inward current or the elevation in [Ca2+](i) induced by 3,5-DHPG. Furthermore, the inward current induced by 3,5-DHPG was neither blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy] ethyl-1H-imidazole, an inhibitor of store operated calcium influx, nor by nimodipine or omega-agatoxin, blockers of voltage-gated calcium channels. These electrophysiological and Ca2+-imaging experiments suggest that the mGluR(1)-mediated inward current, although mainly carried by Na+, involves influx of Ca2+ from the extracellular space.