氢化可的松抑制Jurkat细胞的调节性体积减小

目的 :研究氢化可的松对Jurkat细胞调节性体积减小 (regulatoryvolumedecrease ,RVD)的影响及可能的机制。方法 :通过MoticImagesAdvanced 3.1软件系统实时监测细胞体积的变化 ,并通过3 H掺入实验观察Jurkat细胞增殖。结果 :氢化可的松对Jur kat细胞RVD具有剂量依赖性的抑制作用 ,在 10 -4和 10 -3 mol·L-1时可以明显抑制Jurkat细胞的RVD ,而在 10 -9、10 -7和 10 -5mol·L-1时对Jurkat细胞的RVD没有明显影响。钾通道阻断剂奎宁 (quinine)亦可以抑制Jurkat细胞的RVD。氢化可的松和奎宁在10 -3 mol·L-1均可明显抑制Jurkat的增殖 (包括自然增殖和ConA诱导增殖 )。结论 :氢化可的松对Jur kat细胞RVD的抑制可能是通过钾通道起作用     

KATP开放剂埃他卡林对高K+刺激PC12细胞释放谷氨酸的影响

目的：研究KATP开放剂埃他卡林（iptkalim,IPT)对高K^ 刺激PC12释放谷氨酸（Glu)的影响及其作用机制。方法：以培养的PC12细胞为模型细胞，应用HPLC法测定细胞培养液中Glu含量。结果：IPT以浓度依赖方式抑制高K^ 刺激PC12细胞释放Glu，格列苯脲（Gli)增强高K^ 刺激PC12细胞释放Glu的效应，并部分逆转IPT的抑制效应，PKC抑制剂HA-100和钙调素（CaM)拮抗剂三氟拉嗪不影响高K^ 刺激PC12细胞释放Glu，也不拮抗Gli的增强效应。结论：IPT抑制Glu释放与开放KATP有关，Gli拮抗IPT的抑制效应并非由PKC或CaM信号传导途径介导。     

雷公藤内酯:一种T-淋巴细胞核因子-кB的强效抑制剂(英文)

目的:探讨雷公藤内酯醇对T淋巴细胞中核因子-kB活力及其抑制分子IkBα的影响,以进一步阐明其免疫抑制效应的分子机制.方法:将不同浓度的雷公藤内酯醇处理普通培养状态的和同时使用PMA/PHA激活的Jurkat细胞,以凝胶迁移率实验(EMSA)检测细胞NF-kB活力的改变,并以逆转录-半定量PCR方法检测处理前后细胞中IkBα mRNA水平的改变.结果:(1)在普通培养状态下的Jurkat细胞中存在有一定活力的NF-kB,使用PMA/PHA处理可显著升高Jurkat细胞中NF-kB的活力,雷公藤内酯醇可以降低两种培养状况下的Jurkat细胞中NF-kB活力,并以激活状态下更为显著.(2)雷公藤内酯醇可以上调Jurkat细胞中IkBa mRNA的转录水平.结论:雷公藤内酯醇对T细胞内IkBa基因转录的调控以及抑制T细胞内NF-kB的活力是雷公藤免疫抑制效应的分子机制之一.     

螺内酯诱导人急性白血病细胞Jurkat凋亡的作用研究

目的研究螺内酯对人急性白血病细胞Jurkat体外增殖的抑制及诱导凋亡的作用。方法将终浓度分别是10、50及100μmol/L的螺内酯加入Jurkat细胞的培养体系中,24 h内每隔4 h通过MTT法分析Jurkat细胞的增殖抑制率,Annexin V/PI流式细胞术法分析Jurkat细胞的早期凋亡率。结果螺内酯与Jurkat细胞共同培养12 h后,螺内酯能显著增加对细胞的增殖抑制作用,并且与药物浓度成正相关,各浓度组的抑制率随着培养时间的延长而升高,与药物干预时间成正相关;螺内酯处理Jurkat细胞24 h,各浓度组诱导细胞凋亡率分别为:10μmol/L组(11.2±0.35)%、50μmol/L组(29.8±1.27)%及100μmol/L(56.5±1.41)%,各个浓度组诱导细胞凋亡率与药物浓度成正相关。结论螺内酯对人T淋巴细胞白血病Jurkat细胞具有抑制增殖和诱导凋亡的作用,提示该药可能具有潜在抑瘤作用。     

紫杉醇诱导Jurkat细胞凋亡的实验研究

目的：探讨紫杉醇对Jurkat（人T细胞白血病细胞株）细胞增殖抑制丰及细胞周期各时相变化的影响。方法：应用MTT比色法和流式细胞仪检测紫杉醇对Jurkat细胞增殖抑制的时间效应，浓度效应及凋亡率。结果：紫杉醇对Jurkat细胞有明显的抑制作用，且呈剂量依赖性；流式细胞仪分析：G1期细胞增高，S期细胞减少，G2／M期细胞相对增多。结论：紫杉醇能够抑制Jurkat细胞增殖，抑制GT期细胞向S期的进程，促进Jurkat细胞凋亡。     

香菇多糖对小鼠T淋巴细胞膜上离子通道基因表达的影响

目的:研究香菇多糖对小鼠T淋巴细胞膜上离子通道基因表达的影响。方法:40只KM小鼠随机分为对照(等容生理盐水)组与香菇多糖高、中、低剂量(12.5、2.5、0.5 mg/kg)组,腹腔注射,每天1次,连续30 d。末次给药1 h后处死小鼠,采用实时荧光定量聚合酶链反应(RT-qPCR)法检测小鼠T淋巴细胞内电压门控钾离子通道(KV1.3)、Ca2+激活的钾离子通道(KCa3.1)、阳离子通道(TRPM7)、氯离子通道(Clswell)以及钙离子通道(CRAC)激活的调控元件基质相互作用因子1(STIM1)和组成分子orai1mRNA的表达。结果:与对照组比较,香菇多糖中剂量组小鼠T淋巴细胞KV1.3、KCa3.1、STIM1、orai1、TRPM7、Clswell mRNA表达增强,差异有统计学意义(P<0.05)。结论:香菇多糖可提高小鼠T淋巴细胞增殖及活性,其机制与增强T细胞上的KV1.3、KCa3.1、orai1、STIM1、TRPM7、Clswel mRNA表达有关。     

稳定的人T-T杂交瘤的建立

本文报道一株能产生B细胞生长因子(BCGF)的T-T杂交瘤.实验选择5种耐受6-硫鸟嘌呤或8-氮鸟嘌呤而对氨基喋呤敏感的传代T淋巴样细胞系(CEM TG Ell、CEMF3918、MOLT-4、Jurkat 1和Jurkat F3250)供细胞融合用,用于融合的T淋巴细胞得自正常供体外周血,用10%胎牛血清(FCS)-RPMI配成1×10~6/ml的细胞悬液,加最终浓度为0.75%的植物血凝素(PHA-M),置37℃、5%CO_2中温育24～72小时,继将PHA刺激的正常T细胞与上述传代T细胞按5∶1的比例混合,融合剂用50%PEG3500.在融合5周后     

哇巴因诱导Jurkat细胞凋亡对白细胞介素2受体α亚基的影响

目的探讨哇巴因诱导Jurkat细胞凋亡与白细胞介素2受体α亚基(IL-2Rα)的关联。方法 MTT法检测哇巴因对Jurkat细胞生长的影响;Western blot法检测IL-2Rα的表达;ELISA法检测细胞培养液中可溶性IL-2Rα(sIL-2Rα)的改变。结果低浓度(30、50nM)哇巴因能够诱导Jurkat细胞凋亡,下调Jurkat细胞表面的IL-2Rα及sIL-2Rα表达。结论 IL-2Rα参与了哇巴因诱导Jurkat细胞凋亡的调节。     

脱氢表雄甾酮对COPD大鼠支气管平滑肌细胞钙激活性钾通道的激活作用

目的:对比研究正常及慢性阻塞性肺病(COPD)大鼠支气管平滑肌细胞钙激活性钾(KCa)通道的活性,观察脱氢表雄甾酮(DHEA)对COPD大鼠支气管平滑肌细胞KCa通道的作用。方法:38只雄性SD大鼠随机分为对照组(n=20)和COPD组(n=18),在相对密闭舱内吸入纸烟建立COPD动物模型,采用急性酶分离法分离大鼠支气管平滑肌细胞,应用膜片钳技术,在对称性高钾溶液中,于急性分离的大鼠支气管平滑肌细胞的内面向外式膜片上,分离出KCa电流,比较COPD和对照组的活性,记录DHEA对COPD大鼠支气管平滑肌细胞膜KCa通道活性的激活作用。结果:COPD组KCa活性明显比正常组低(P<0.01),DHEA可明显激活COPD组大鼠支气管平滑肌的KCa电流(P<0.01)。结论:COPD大鼠支气管平滑肌细胞KCa通道活性降低可能在COPD发病机制中起着一定作用,DHEA可以直接激活COPD大鼠支气管平滑肌KCa活性,松驰COPD大鼠支气管平滑肌。     

谷氨酰胺对肿瘤生长和肝癌细胞凋亡的影响(英文)

目的:探索谷氨酰胺对肿瘤生长及肝癌细胞凋亡的影响。方法:在小鼠右腋皮下接种H22肿瘤细胞悬液,灌服含谷氨酰胺(GLN)液;在人肝癌细胞培养液中加入不同浓度的GLN液。分别检测小鼠血浆及细胞培养液中MDA、NO及细胞中GSH含量,观察小鼠右腋皮下肿瘤生长及肝癌细胞增殖和凋亡情况。结果:灌服GLN液,有抑制皮下肿瘤块生长的作用;在人肝癌细胞培养液中加入一定浓度的GLN液,有抑制肝癌细胞增殖的作用并促使肝癌细胞凋亡。同时小鼠血浆和细胞培养液中NO含量升高,MDA稍有下降;细胞粉碎液中,GSH升高。结论:GLN对肿瘤生长的抑制作用及对肝癌细胞凋亡的影响可能与使抗氧化活性的提高、阻抑自由基对癌细胞增殖的介导及促使NO释放有关。     

Calcium-activated potassium channels in vasculature in response to ischemia-reperfusion

Based on the genetic relationship, single-channel conductance, and gating mechanisms, calcium-activated potassium (KCa) channels identified in vasculature can be divided into 3 groups including large-conductance KCa, small, and intermediate conductance KCa. KCa channels in smooth muscle and endothelial cells are essential for the regulation of vascular tone. Vascular dysfunction under ischemia-reperfusion (I-R) or hypoxia-reoxygenation (H-R) conditions is associated with modulations of KCa channels that are attributable to multiple mechanisms. Most studies in this regard relied on the change of relaxation components sensitive to certain channel blockers to indicate the alteration of KCa channels under I-R conditions, which however provided conflicting results for the effect of I-R. The possible mechanisms involved in KCa channel modulation under I-R/H-R include overproduction of reactive oxygen species such as superoxide anion, hydrogen peroxide, and peroxynitrite, increase of intracellular H ion, and lactate accumulation, etc. However, more studies are necessary to further understand the discrepancies in the sensitivity of KCa channels to I-R injury in different vascular beds.     

Uncoupling of K+ and Cl- transport across the cell membrane in the process of regulatory volume decrease

It is accepted that K(+) and Cl(-) flows are coupled tightly in regulatory volume decrease (RVD). However, using self referencing microelectrodes, we proved that K(+) and Cl(-) transport mainly by channels in RVD was uncoupled in nasopharyngeal carcinoma CNE-2Z cells, with the transient K(+) efflux activated earlier and sustained Cl(-) efflux activated later. Hypotonic challenges decreased intracellular pH (pH(i)), and activated a proton pump-dependent H(+) efflux, resulting in a decline of extracellular pH (pH(o)). Modest decreases of pH(o) inhibited the volume-activated K(+) outflow and RVD, but not the Cl(-) outflow, while inhibition of H(+) efflux or increase of pH(o) buffer ability promoted K(+) efflux and RVD. The results suggest that the temporal dynamics of K(+) channel activities is different from that of Cl(-) channels in RVD, due to differential sensitivity of K(+) and Cl(-) channels to pH(o). H(+) efflux may play important roles in cell volume regulation, and may be a therapeutic target for human nasopharyngeal carcinoma.     

Inwardly rectifying potassium channels in the regulation of vascular tone

Potassium ion (K+) channel activity is one of the major determinants of vascular muscle cell membrane potential and thus vascular tone. Four types of K+ channels are functionally important in the vasculature-Ca(2+)-activated K+ (KCa) channels, voltage-dependent K+ (Kv) channels, ATP-sensitive K+ (KATP) channels, and inwardly rectifying K+ (KIR) channels, and the latter type will be the subject of this review. Recent advances in vascular KIR channel research indicate that this channel: 1) is present in vascular muscle; 2) modulates basal arterial tone; 3) mediates powerful hyperpolarization and vasodilator responses to small but physiological increases in extracellular K+; 4) may contribute to vasodilatation in response to flow-induced shear stress; 5) may be inhibited by protein kinase C activity; 6) may be involved in vasorelaxation mediated by endothelium-derived hyperpolarizing factor; and 7) may be functionally altered in cardiovascular diseases. Vascular effects of KIR channels have so far been most extensively studied in the cerebral circulation where KIR function may be important in coupling cerebral metabolism and blood flow.     

Involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of porcine coronary artery endothelial cells

This study investigated the effects of the following adenosine agonists: 5;-ethylcarboxamidoadenosine (NECA), N6-cyclopentyadenosine (CPA) 2-[p-(2-carboxyethyl)]phenylamino-5;N-ethylcarboxamidoadenosine (CGS-21680), and 2-chloroadenosine (CAD) and its antagonist, 4-(2-[7-amino-2-[2-furyl]]1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385), a selective A2A adenosine receptor antagonist, and the involvement of the K+ATP and KCa channels on the resting membrane potential (RMP) of confluent monolayers of cultured porcine coronary artery endothelial cells (PCAECs). Adenosine agonists and K+ATP channel openers (pinacidil, cromakalim) hyperpolarized cultured PCAECs. The average RMP was -32.31 +/- 1.2 mV. Adenosine agonists at 10-5 M caused a significant increase in RMP to -65.0 +/- 1.5 mV for CAD (a nonselective adenosine receptor agonist) to -75.9 +/- 1.6 mV for CGS-21680 (a selective A2A receptor agonist) and to -87.0 +/- 3.5 mV for NECA (a nonselective A1/A2A/A2B receptor agonist). Pinacidil and cromakalim at 10 microM increased the membrane potential to -76.2 +/- 1.2 mV and -75.22 +/- 0.12 mV, respectively. The hyperpolarization induced by adenosine receptor agonists and KATP openers was inhibited by an application of the K+ATP channel blocker glibenclamide (10 microM), indicating the involvement of the K+ATP channel in the adenosine-mediated hyperpolarization of PCAECs. Moreover, 1-EB10, a selective opener of the maxi-KCa channel, hyperpolarized PCAECs, and the effect of 1-EB10 was completely blocked by a selective, irreversible blocker of the high conductance KCa (maxi-K) channels (penitrem A), but it only partially blocked the effect of NECA. ZM-241385 has no effect on hyperpolarization elicited by K+ATP and KCa channel openers. However, ZM-241385 significantly blocked the hyperpolarization effect of CAD and CGS-21680. ZM-241385 partially blocked the hyperpolarizing effect of NECA, and a combination of ZM-241385 and penitrem A further blocked the hyperpolarizing effect of NECA. These results further support the involvement of K+ channels in adenosine A2A and A2B receptor-mediated hyperpolarization of PCAECs.     

猪脾脏中的应激免疫抑制蛋白类似物(英文)

目的:研究猪脾脏中的一种具有免疫抑制活性的蛋白质,纯化该蛋白并研究其与临床疾病的关系。方法:1)稀酸提取;2)超滤透析分离分子量大于M_r30 000的分子;3)FPLC免疫亲和层析纯化;4)T淋巴细胞转化及ELISA检测其功能活性;5)SDS-PAGE检测其分子量及纯度。结果:该蛋白能抑制由Con A诱导的淋巴细胞增殖。其分子量在190 000 左右。该蛋白对某些来源于免疫细胞系的细胞,具有明显的选择性抑制作用。结论:成功部分纯化这种蛋白,并证明其与应激免疫抑制蛋白有相似的理化、免疫原性和功能活性。这一研究为进一步大量纯化该蛋白提供了实用和稳定的生物学来源,并为研究它与肿瘤、自身免疫性等临床疾病关系开辟了一条新的研究途径。     

Effect of plant flavonoids on the volume regulation of rat thymocytes under hypoosmotic stress

BackgroundCell volume regulation and volume-regulated anion channels are critical for cell survival in non-isosmotic conditions, and dysregulation of this system is detrimental. Although genes and proteins underlying this basic cellular machinery were recently identified, the pharmacology remains poorly explored.MethodsWe examined effects of 16 flavonoids on the regulatory volume decrease (RVD) of thymocytes under hypoosmotic stress assessed by light transmittance and on the activity of volume-sensitive chloride channel by patch-clamp technique.ResultsComparison of effects of flavonoids on RVD revealed a group of four active substances with lehmannin being the strongest inhibitor (IC₅₀ = 8.8 μM). Structure-functional comparison suggested that hydrophobicity brought about by methoxy, prenyl or lavandulyl groups as well as by the absence of glucosyl fragment together with localization of the phenyl ring B at the position C2 (which is at C3 in totally inactive isoflavones) are important structural determinants for the flavonoids activity as volume regulation inhibitors. All active flavonoids suppressed RVD under Gramicidin D-NMDG hypotonic stress conditions when cationic permeability was increased by an ionophore, gramicidin D, with all extracellular monovalent cations replaced with bulky NMDG⁺ suggesting that they target volume-sensitive anionic permeability. While effects of hispidulin and pulicarin were only partial, lehmannin and pinocembrin completely abolished RVD under Gramicidin D-NMDG conditions. In direct patch-clamp experiments, lehmannin and pinocembrin produced a strong inhibiting effect on the swelling-induced whole-cell chloride conductance in a voltage-independent manner.ConclusionLehmannin, pinocembrin, and possibly hispidulin and pulicarin may serve as leads for developing effective low-toxic immunomodulators.     

The small molecule NS11021 is a potent and specific activator of Ca2+-activated big-conductance K+ channels

Large-conductance Ca(2+)- and voltage-activated K(+) channels (Kca1.1/BK/MaxiK) are widely expressed ion channels. They provide a Ca(2+)-dependent feedback mechanism for the regulation of various body functions such as blood flow, neurotransmitter release, uresis, and immunity. In addition, a mitochondrial K(+) channel with KCa1.1-resembling properties has been found in the heart, where it may be involved in regulation of energy consumption. In the present study, the effect of a novel NeuroSearch compound, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea (NS11021), was investigated on cloned KCa1.1 expressed in Xenopus laevis oocytes and mammalian cells using electrophysiological methods. NS11021 at concentrations above 0.3 microM activated KCa1.1 in a concentration-dependent manner by parallel-shifting the channel activation curves to more negative potentials. Single-channel analysis revealed that NS11021 increased the open probability of the channel by altering gating kinetics without affecting the single-channel conductance. NS11021 (10 microM) influenced neither a number of cloned Kv channels nor endogenous Na(+) and Ca(2+) channels (L- and T-type) in guinea pig cardiac myocytes. In conclusion, NS11021 is a novel KCa1.1 channel activator with better specificity and a 10 times higher potency compared with the most broadly applied KCa1.1 opener, NS1619. Thus, NS11021 might be a valuable tool compound when addressing the physiological and pathophysiological roles of KCa1.1 channels.     

Blockade of Ca2+ -activated K+ channels by galantamine can also contribute to the potentiation of catecholamine secretion from chromaffin cells

Galantamine is a drug in clinical use for the treatment of Alzheimer's disease, but its mechanism(s) of action remains controversial. Here we addressed the question whether galantamine could potentiate neurotransmitter release by inhibiting small conductance Ca2+ -activated K+ channels (KCa2). Galantamine potentiated catecholamine secretory responses induced by 10 s pulses of acetylcholine and high [K+]o applied to fast-superfused bovine adrenal chromaffin cell populations. Catecholamine release was significantly enhanced by galantamine although we did not find concentration dependence in the range 0.1-1 microM. The KCa2 channel blocker apamin (0.3 microM) occluded the potentiating effects of galantamine on acetylcholine-evoked secretion. Like apamin, galantamine also modified the firing of action potentials, but to a lesser extent. In addition, 1 microM galantamine reduced by 41% the KCa2 current without modifying the voltage-dependent Ca2+ currents. These results constitute the first direct evidence that galantamine can potentiate neurotransmitter release by blocking KCa2 channels, in addition to its already demonstrated capacity to mildly block acetylcholinesterase or potentiate allosterically nicotinic receptors.     

Hypoxic stress induces transient receptor potential melastatin 2 (TRPM2) channel expression in adult rat cardiac fibroblasts

When cardiac tissue is exposed to hypoxia, myocytes are damaged, while fibroblasts are activated. However, it is unknown what changes are induced by hypoxia in cardiac fibroblasts. In this study, using the whole cell patch-clamp technique, we investigated the effect of hypoxia on membrane currents in fibroblasts primarily cultured from adult rat hearts. Cardiac fibroblasts were incubated for 24 h under normoxic or hypoxic conditions using Anaeropack. Hypoxia increased a current which reversed at around -20 mV in the cardiac fibroblasts. This current was inhibited by clotrimazole, which is an inhibitor of transient receptor potential melastatin 2 (TRPM2) channel and intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1). ADP ribose in the pipette solution enhanced this current. Quantitative RT-PCR revealed that mRNA of TRPM2, but not that of KCa3.1, was increased by hypoxia. RNA interference of TRPM2 prevented the development of the hypoxia-induced current. H(2)O(2), an activator of TRPM2 channel, induced a higher [Ca(2+)](i) elevation in hypoxia-exposed cardiac fibroblasts than that in normoxia-exposed cells. We conclude that hypoxia induces TRPM2 channel expression in adult rat cardiac fibroblasts.     

K(Ca) channel-opening activity of Ginkgo Biloba extracts and ginsenosides in cultured endothelial cells

1. Extracts of Ginkgo biloba (EGb) and ginsenosides (GS) have been reported to induce vasorelaxation. In the present study, the role of K+ channels in the action of EGb and GS to activate nitric oxide synthase (NOS) activity was investigated in cultured endothelial cells. 2. Nitric oxide synthase activity of cultured endothelial cells detected by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) histochemistry method was significantly increased after treatment with 20 microg/mL EGb or 40 microg/mL GS plus 10 mmol/L L-arginine. The effect was completely abolished by the addition of 0.5 micromol/L Nomega-nitro-L-arginine, an inhibitor of NOS, to the incubation medium and partially inhibited by 10 micromol/L tetraethylammonium (TEA), an inhibitor of Ca2+-activated K+ (KCa) channels. 3. Application of EGb to the intracellular surface of excised inside-out patches activated K+ channels in a concentration-dependent manner in the concentration range 1-100 microg/mL. Channel activity was also activated by application of GS at concentrations ranging from 1 to 300 microg/mL. The modulation of channel activity was inhibited by 0.5 mmol/L TEA but not by 0.5 mmol/L glibenclamide, an inhibitor of ATP-sensitive K+ channels. 4. Thus, in cultured endothelial cells, the increase in NOS activity induced by EGb or GS depends on the activity of KCa channels. These compounds may regulate nitric oxide release by changing the cell membrane potential in vascular endothelial cells.