水飞蓟宾-磷脂酰胆碱复合物诱导HepG2细胞凋亡研究

目的研究水飞蓟宾-磷脂酰胆碱复合物（Silybin-phosphatidylcholine Compound,SPC）诱导肝癌HepG2细胞凋亡作用及其机制。方法不同浓度SPC处理肝癌HepG2细胞,通过MTT法、细胞形态学观察、流式细胞仪和激光共聚焦显微镜观察SPC对HepG2细胞的生长抑制作用及其细胞凋亡的影响。结果SPC可抑制HepG2细胞的增殖。其GI50为46．8μg/ml;50μg／ml的SPC作用HepG2细胞24h后,细胞出现早期调亡的形态;75μg／ml、100μg／ml的SPC对HepG2细胞凋亡率分别为（17．22±3．45）％和（25．50±5．72）％;50μg／ml、75μg／ml、100μg／ml的SPC作用于HepG2细胞24h后,细胞内的Ca^2＋浓度显著升高（P〈0．05,P〈0．01）。结论SPC能够诱导肝癌细胞系HepG2细胞凋亡,其机制与SPC升高细胞内Ca^2＋浓度有关。     

单纯疱疹病毒1型诱导的Hela细胞凋亡及钙浓度的变化

一方面认为宿主细胞可利用细胞凋亡来清除病毒感染细胞 ;另一方面病毒可以抑制细胞凋亡 ,以利于自身增殖需要。而病毒诱导细胞凋亡还是抑制凋亡 ,取决于多方面因素。Ｌｅｐｐａｒｄｉ研究表明野生株ＨＳＶ 1感染Ｖｅｒｏ细胞并不引起Ｖｅｒｏ细胞凋亡 ,但缺乏调节性基因α4的Ｈ     

细胞内pH,钙离子与细胞凋亡

细胞凋亡与细胞内ｐＨ（ｐＨｉ）、细胞内钙离子浓度（［Ｃａ２＋］ｉ）密切相关，不同的刺激信号通过复杂的信息传递途径引起ｐＨｉ和［Ｃａ２＋］ｉ的变化，细胞内酸化或［Ｃａ２＋］ｉ的增高并伴有细胞内碱化分别是ＤＮａｓｅⅡ或ＤＮａｓｅⅠ激活的必要条件，但单独的细胞内碱化并不能激活ＤＮａｓｅⅠ。核酸内切酶的激活及其催化的基因组ＤＮＡ在核小体间降解是细胞凋亡的特征性生化事件。而Ｎａ＋／Ｈ＋交换蛋白－１（ＮＨＥ－１）的失活可能在诱导凋亡中起关键作用。     

松针油诱导宫颈癌Hela细胞凋亡及其作用机制研究

目的 研究松针油对人宫颈癌Hela细胞生长的抑制作用，探讨Hela细胞发生凋亡的分子机制。方法 采用水蒸气蒸馏法提取松针油，气相色谱一质谱联用仪分析成分。MTT法测定松针油对Hela细胞增殖的影响。流式细胞仪和Hochest 33258检测药物作用前后细胞凋亡的情况。应用Western blotting观察Hela细胞caspase-3酶原的变化。用caspase活性检测试剂盒检测caspase-3，8，9的活性。结果 MTT结果显示松针油对Hela细胞生长有明显的抑制作用，呈显著的时间和浓度依赖性。流式细胞仪分析显示松针油诱导Hela细胞发生凋亡，细胞表现出典型的凋亡特征，并且可见凋亡小体，且凋亡率具有时间依赖性。松针油诱导Hela细胞凋亡过程中caspase．3被激活。Caspase-8，9活性增高，又有时间依赖性，且有时间顺序。结论 松针油对Hela细胞的生长有明显的抑制作用，其作用机制是松针油可诱导Hela细胞发生凋亡。在诱导细胞凋亡的过程中，caspase-3的激活是介导Hela细胞发生凋亡的共同通路。同时，活化的caspase-8，9可能参与了caspase-3的激活，从而诱导凋亡的发生。     

长春新碱诱导的自噬性凋亡与细胞内游离Ca^2＋浓度的相关性研究

目的 研究长春新碱（ＶＣＲ）诱导的Ｌ－０２细胞自噬性凋亡细胞内游离钙离子浓度（［Ｃａ＾２＋］ｉ）的变化,以及自噬特异性抑制剂３－ｍｅｔｈｙｌａｄｅｎｉｎｅ（３ＭＡ）对此自噬性凋亡和［Ｃａ＾２＋］ｉ的影响。方法 应用已建立的ＶＣＲ诱导的Ｌ－０２细胞自噬性凋亡模型,使用电镜、流式细胞术检测细胞;用Ｆｌｕｏ－３／ＡＭ荧光探针经流式细胞仪测定Ｌ－０２细胞平均［Ｃａ＾２＋］ｉ。结果 电镜及流式细胞术检测证实     

TRAIL诱导Hela细胞凋亡的线粒体信号通路

 目的 探讨 TRAIL 诱导人宫颈癌 Hela 细胞凋亡的线粒体通路。 方法 琼脂糖凝胶电泳判断细胞凋亡;激光共聚焦、Western blot、荧光免疫和 caspase-3 活性检测测定细胞线粒体膜电位 (∆Ψm) 、Bcl-2 蛋白、细胞色素 c (Cyt c) 和凋亡诱导因子 (AIF) 蛋白在细胞中的定位以及 caspase-3 活性。结果 TRAIL 能诱导 Hela 细胞凋亡,有凋亡细胞特有的 DNA 梯状条带。同时,TRAIL具有时间依赖性致 ∆Ψm 和 Bcl-2 蛋白含量明显下降,线粒体 Cyt c 蛋白释放,AIF 蛋白向细胞质、细胞核转移,caspase-3 活性增强。结论 TRAIL 诱导 Hela 细胞凋亡途径之一是通过线粒体信号通路进行的。     

Bak基因对Hela细胞的细胞周期和细胞凋亡的调控作用

目的 研究Ｂｃ１－２基因家族促凋亡蛋白Ｂａｋ在细胞凋亡细胞周期调控中的作用,评价它们作为肿瘤治疗的潜在靶基因的可能性。方法 采用一种可诱导性表达系统,（ＭＴ－Ⅱ调节系统）,在外加锌离子（ＺｎＳＯ４,１００μｍｏｌ／Ｌ）的条件下诱导Ｂａｋ基因表达。以Ｈｅｌａ细胞系作为靶细胞,获得表达Ｂａｋ基因的稳定转染子。结果 可诱导性Ｂａｋ基因过表达的Ｈｅｌａ细胞出现广泛死亡。ＴＵＮＥＬ染色证实细胞核碎片化,提示     

问号钩端螺旋体内化过程中细胞内游离Ca2+水平变化及细胞凋亡

目的建立观察问号钩端螺旋体(简称钩体)黏附的双荧光染色法,探讨不同毒力钩体对细胞内游离Ca2+水平及细胞凋亡的影响.方法以问号钩体黄疸出血群赖型56601株和双曲钩体三堡垄群patoc型PatocⅠ株抗血清为一抗、羊抗兔IgG荧光素F(ab)2和罗丹明F(ab)2片段为二抗的双荧光染色法,分别检测56601株和PatocⅠ株钩体对Vero、J774A.1细胞的黏附作用.采用fluo-3/AM胞内Ca2+特异荧光标记激光共聚焦技术,检测问号钩体56601株和波摩那群波摩那型56608株、双曲钩体PatocⅠ株作用的J774A.1细胞胞内游离Ca2+水平的变化.采用FITC-annexinⅤ/PI荧光标记流式细胞术,检测紫外线灭活前后的56601株钩体诱导Vero和J774A.1细胞凋亡的情况.结果所建立的双荧光染色法能清晰地观察到强毒力的56601株钩体对Vero和J774A.1细胞的黏附,无毒力的PatocⅠ株钩体则否.正常J774A.1细胞胞内游离Ca2+基础值为(105.0±7.0)%,PatocⅠ株钩体作用细胞的荧光强度变化百分数一直波动于(102.2±5.2)%.56601株钩体感染J774A.1细胞胞内游离Ca2+浓度迅速增高,呈现为双峰型曲线,其荧光强度变化百分数分别为(747.5±35.7)%和(804.6±40.8)%.56608株钩体感染J774A.1细胞胞内游离Ca2+浓度呈现为缓慢的单一坡型升高,其最大荧光强度变化百分数为(402.4±23.6)%,明显小于56601株钩体,差异有统计学意义(P<0.01).紫外线灭活前后56601株钩体作用Vero细胞的凋亡率分别为84.5%和78.2%,J774A.1细胞凋亡率分别为34.5%和30.9%.结论所建立的双荧光染色法可用于观察钩体的黏附.细胞胞内游离Ca2+水平与所感染的钩体菌株毒力成正相关.有毒力的钩体接触细胞时即可诱导凋亡发生,启动细胞凋亡信号通路的配体分子可能位于钩体表面.     

透骨草提取物诱导人宫颈癌Hela细胞凋亡及其相关机制研究

目的:探讨透骨草提取物诱导人宫颈癌Hela细胞凋亡及其相关机制。方法:荧光显微镜观察Hela细胞形态,透射电镜观察Hela细胞超微结构,流式细胞术检测Hela细胞凋亡率,Western blot检测Bcl-2蛋白和Caspase-3蛋白表达水平。结果:100μg/ml透骨草提取物处理的Hela细胞后,荧光显微镜显示Hela细胞皱缩,细胞核浓缩呈新月状边集。透射电镜可见Hella细胞表面突起和微绒毛减少,核断裂、染色质凝聚且边缘化,有"出芽"现象。流式细胞术显示透骨草提取物处理的Hela细胞凋亡率为(25.90±1.13)%,明显高于对照组(P<0.01)。Western blot结果显示透骨草提取物处理的Hela细胞Bcl-2蛋白和Caspase-3蛋白表达达明显低于与对照组(P<0.05)。结论:透骨草提取物可诱导Hela细胞凋亡,其机制可能与下调Bcl-2蛋白和Caspase-3蛋白表达有关。     

乙酰唑胺对水孔蛋白-1表达的抑制与细胞内pH及Ca2+的关系

为明确乙酰唑胺对水孔蛋白-1(aquaporin-1,AQP1)基因表达的抑制是否与其对细胞内pH和Ca2+浓度的影响有关,采用激光共聚焦扫描显微镜的技术观察不同培养条件下(培养液中含或不含NaHCO3),10-5 mol*L-1乙酰唑胺对原代培养的大鼠肾脏近曲小管上皮细胞内pH和Ca2+浓度的影响,同时用免疫荧光法观察给药1d、3d、7d后AQP1表达的变化.结果显示在含NaHCO3组,乙酰唑胺处理7d后,使细胞内的H+探针的荧光强度由91.81±5.44降至39.58±3.10, Ca2+探针荧光强度由18.23±1.02降至11.5±1.03,AQP1蛋白荧光强度由44.4±1.86减少到25.91±1.97;在不含NaHCO3组,细胞内pH和Ca2+浓度均无明显变化;但AQP1蛋白的荧光强度由43.15±2.97明显减少到23.85±1.92.结果表明乙酰唑胺对细胞内pH和Ca2+浓度的调节依赖于细胞外液HCO3-的存在,而它对AQP1表达的抑制与细胞内pH和Ca2+浓度的改变无关.     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na+/Ca2+ exchange mechanism

Törnquist , K. 1992. Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na⁺/Ca²⁺ exchange mechanism. Acta Physzol Scand 144 , 341–348. Received 28 April 1 991 , accepted 30 October 1991. ISSN 0001–6772. Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland. The effect of extracellular Na⁺ on cytosolic free Ca²⁺ and on influx and efflux of Ca²⁺ was investigated in FRTL-5 thyroid cells. Stimulating the cells with the purinergic agonist ATP induced a rapid efflux of ⁴⁵Ca²⁺ from cells loaded with ^4aCa²⁺. Replacement of extracellular Na⁺ with choline⁺, significantly decreased the adenosine triphosphate-induced efflux of ⁴⁵Ca²⁺. Furthermore, adenosine triphosphate-induced uptake of ⁴⁵Ca²⁺ was increased when extracellular Na⁺ was replaced with choline⁺, compared with the uptake seen in Na⁺ buffer. Replacing extracellular Na⁺ with choline⁺, increased resting levels of cytosolic free Ca²⁺ from 50 ± 2 nM (mean ± SE) to 81 ± 3 nM (P < 0.05) in Fura 2 loaded cells. In cells preincubated with 1 mM ouabain for 30 min, resting cytosolic free Ca²⁺ increased to 73 ± 3 nM (P < 0.05). In a Na⁺ buffer, the adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ was 872 ± 59 nM, compared with 1070 ±63 nM in choline' buffer (P < 0.05). The plateau level of cytosolic free Ca²⁺ in response to adenosine triphosphate was 130±16 nM in Na⁺ buffer, compared with 209±9 nM in choline⁺ buffer (P < 0.05). Readdition of Na⁺ to the plateau phase decreased cytosolic free Ca⁺² to 152 ± 5 nM. Stimulating the cells with 10 μM of the Na^+-selective monovalent ionophore monensin increased cytosolic free Ca²⁺ from 53 ± 9 nM to 12416 nM (P < 0.05). This increase in cytosolic free Ca²⁺ was dependent on both extracellular Na⁺ and extracellular Ca²⁺ If cells were first stimulated with monensin, and then with adenosine triphosphate, the transient increase in cytosolic free Ca²⁺ was 1027 ± 24 nM (P < 0. 05 , compared with control cells). The results thus indicate, that FRTL-5 cells have a functional Na⁺/Ca²⁺ exchange mechanism and that this mechanism is of importance in restoring adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ to resting cytosolic free Ca²⁺ levels.     

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

 The influence of intracellular pH (pH_i) on intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells was examined microspectrofluorometrically. pH_i was changed by replacing phosphate buffer by the diffusible buffers CO₂/HCO₃ ^–or NH₃/NH₄ ⁺ (pH 7.4). CO₂/HCO₃ ^–buffers at 2,5 or 10% acidified pH_i by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca²⁺]_i by 8–15 nmol/l. This effect was independent of the extracellular Ca²⁺ activity and the filling state of thapsigargin-sensitive Ca²⁺ stores. Removing the CO₂/HCO₃ ^–buffer alkalinized pH_i by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca²⁺]_i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca²⁺-free solution and with thapsigargin showed that the [Ca²⁺]_i transient was due to release from intracellular pools and stimulated Ca²⁺ entry. NH₃/NH₄ ⁺ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca²⁺]_i to a peak (Δ [Ca²⁺]_i = 164 nmol/l). The peak [Ca²⁺]_i increase was not influenced by removal of external Ca²⁺, but the decline to basal [Ca²⁺]_i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP ₃) antagonist theophylline had any influence on the NH₃/NH₄ ⁺-stimulated [Ca²⁺]_i increase, whereas carbachol-induced [Ca²⁺]_i transients were reduced by more than 80% and 30%, respectively. InsP ₃ measurements showed no change of InsP ₃ during exposure to NH₃/NH₄ ⁺, whereas carbachol enhanced the InsP ₃ concentration, and this effect was abolished by U73122. The pH_i influence on ”capacitative” Ca²⁺ influx was also examined. An acid pH_i attenuated, and an alkaline pH_i enhanced, carbachol- and thapsigargin-induced [Ca²⁺]_i influx. We conclude that: (1) an alkaline pH_i releases Ca²⁺ from InsP ₃-dependent intracellular stores; (2) the store release is InsP ₃ independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca²⁺ influx; (4) the capacitative Ca²⁺ influx activated by InsP ₃ agonists is decreased by acidic and enhanced by alkaline pH_i. The effects of pH_i on [Ca²⁺]_i should be of relevance under many physiological conditions. Received: 17 June 1996 / Received after revision and accepted: 30 August 1996     

Ca2+调控curdlan诱导的巨噬细胞炎症反应中活性氧的生成

目的 观察curdlan刺激下巨噬细胞活性氧(ROS)生成的变化及Ca2对活性氧生成的影响,探讨Ca2+参与curdlan诱导的巨噬细胞炎症反应的可能机制.方法 将Dectin-1缺失的小鼠和正对照WT小鼠来源的巨噬细胞,以100 μg/mL curdlan处理从上述小鼠中分离培养的巨噬细胞,建立巨噬细胞炎症反应模型.处理后,Amplex Red和luminol法分别检测巨噬细胞中超氧离子和过氧化氢的生成变化.用EGTA螯合检测试剂中Ca2+,观察无Ca2情况下活性氧的产生情况,以及在检测试剂中加入不同浓度的Ca2+观察其对ROS产生的影响.结果 Curdlan刺激下,巨噬细胞(骨髓来源和腹腔来源巨噬细胞)中活性氧(包括超氧离子和过氧化氢)水平显著增加.与无Ca2组相比(0 mmol/L),随着Ca2+浓度增加(1.5、2.5 mmol/L),curdlan诱导的巨噬细胞中ROS水平升高.结论 Curdlan能够结合巨噬细胞中相应的免疫识别受体(dectin-1)通过产生大量ROS从而介导炎症反应,调控ROS的水平可能是Ca2+参与炎症反应的机制之一.     

Adaptive control of intracellular Ca2+ release in C2C12 mouse myotubes

Laser scanning confocal imaging was used to monitor release of Ca²⁺ from localized regions in a skeletal muscle cell line with sparsely distributed Ca²⁺ release sites. The goal was to distinguish between two schemes proposed to explain the phenomenon of “quantal” Ca²⁺ release from caffeine-sensitive Ca²⁺ stores in muscle and other tissues: (1) all-or-none (true quantal) Ca²⁺ release from functionally discrete stores that have different sensitivities to caffeine; or (2) adaptive behavior of individual release sites, each responding transiently and repeatedly to incremental caffeine doses. Our results showed that Ca²⁺ release induced by K⁺ or caffeine occurs in discrete loci within the cell. The image areas and fluorescence intensities of some of these evoked local signals were similar to those of Ca²⁺ sparks that were observed under resting conditions and which are believed to be due to spontaneous activation of single release units. In contrast to the expectations imposed by quantal models, incremental doses of caffeine activated the same sets of release sites throughout the cell. Ca²⁺ release, at a given site, triggered by a submaximal dose of caffeine was transient and could be reactivated by addition of a higher caffeine dose, showing the same type of adaptive behavior as measured globally from larger areas of the cell. These results suggest that incremental Ca²⁺ release is accounted for by adaptive behavior of individual Ca²⁺ release sites. Received: 9 August 1995/Received in revised form and accepted: 13 October 1995     

铁离子对Caco-2细胞内钙离子转运的影响及钙离子浓度变化与细胞凋亡关系的研究

目的研究细胞外铁离子(Fe^3 )浓度变化对钙离子(Caco-22)细胞内Ca^2 转运的影响及细胞内钙离子浓度升高与Caco-2细胞凋亡的关系。方法采用激光共聚焦扫描显微镜和流式细胞技术进行观察与检测。结果(1)利用激光共聚焦扫描显微镜(cLSM)观察发现,随着Caco-2细胞外Fe^3 浓度的减少(DFO终浓度为1130～300μmol／L),Ca^2 向细胞内的转运量增加;而随着细胞外Fe^3 浓度的增加(FAC终浓度为10～100μmol／L),Ca^2 向细胞内的转运呈降低趋势;(2)经A23187,和Fluo-3,AM处理后Caco-2细胞的存活率较高,达90％以上(用荧光素二醋酸酯检测);(3)A23187升高Caco-2细胞的胞内Ca^2 浓度,呈剂量依赖性;经流式细胞技术(FCM)检测发现胞内Ca^2 浓度增加具有诱导Caco-2细胞凋亡的作用。结论Caco-2细胞外Fe^3 浓度的减少有促进Ca^2 向细胞内转运的作用。但胞外Fe^3 浓度增加时有抑制Ca^2 向胞内转运的作用;胞内Ca^2 浓度增加对Caco-2细胞凋亡具有诱导作用。     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

羧胺三唑通过抑制NFAT2核转运降低小鼠CD8^+T细胞中程序性死亡受体1的表达

目的研究羧胺三唑(CAI)对CD8+T细胞的作用,并探索其增强活化的T细胞杀伤作用的关键机制。方法用免疫磁珠分选出小鼠CD8+T细胞,分为对照组、CAI组、ZK756326(Ca2+激活剂)组以及CAI+ZK756326组。流式细胞计量术检测细胞内游离钙离子水平;酶联免疫吸附法检测细胞内钙调磷酸酶(CaN)的表达;免疫荧光染色检测细胞活化T细胞核因子2(NFAT2)核转运;染色质免疫共沉淀-qPCR检测细胞中NFAT2调控程序性死亡受体1(PD-1)表达。分离小鼠脾脏细胞毒性T淋巴细胞(CTLs),流式细胞计量术检测其中CD8+T细胞PD-1的表达。结果CAI组显著降低CD8+T细胞内Ca2+浓度(P<0.001),CAI+ZK756326组胞内Ca2+浓度有所升高(P<0.01);CAI显著降低CD8+T细胞中钙调磷酸酶的含量(P<0.001);CAI可显著抑制NFAT2核转运(P<0.001),并使NFAT2依赖的PD-1转录进程显著降低(P<0.001);CAI使小鼠脾脏CTLs细胞中PD-1+CD8+T细胞比例显著减少(P<0.001)。结论CAI通过抑制CD8+T细胞内钙离子水平以及钙调磷酸酶的表达抑制NFAT2核转运,从而降低CD8+T细胞PD-1的表达,进一步产生免疫治疗干预作用。