离子霉素对Jurkat T细胞增殖的抑制作用

目的:探讨离子霉素对Jurkat T细胞增殖的抑制作用.方法:以JurkatT细胞为模型,与5种细胞增殖抑制剂比较,观察离子霉素对JurkatT细胞集落形成的影响,分析不同浓度离子霉素对Jurkat T细胞增殖的影响;分析离子霉素对Jurkat T细胞周期的影响;分析离子霉素对Jurkat T细胞凋亡的作用.本实验结果用统计软件包SPSS10.0进行处理,数据以均数±标准差(x±s)表示,多组间比较采用One-Way ANOVA,两组间比较用非配对Student'st检验.结果:随着离子霉素浓度从0.1 mg/L逐渐增至4.0mg/L,Jurkat T细胞的增殖逐渐减弱,以4.0 mg/L离子霉素的抑制作用最为明显,呈剂量依赖关系,且该浓度下,离子霉素对JurkatT细胞增殖的抑制作用较PD98059、AG490、GF109203X和Genistein明显.离子霉素可使JurkatT细胞周期停滞于G0/G1期,阻止其进入S期并可促进Jurkat T细胞的凋亡.结论:离子霉素对Jurkat T细胞增殖有明显的抑制作用,可使Jurkat T细胞周期停滞于G0/G1期,阻止其进入S期,且对Jurkat T细胞凋亡有显著促进作用.     

硫化砷对Jurkat细胞增殖的抑制作用及其机制

目的：研究硫化砷（As4S4）作用于人类急性T细胞白血病细胞株Jurkat细胞后对其增殖的影响及机制。方法：用不同浓度硫化砷（0,2．5,5,10,20txM）作用于Jurkat细胞不同时间（24,48,72小时）,通过四甲基偶氮唑蓝（MTT）法观察对其增殖的抑制作用。倒置显微镜下观察不同浓度硫化砷（0,5,10,20txM）作用Jurkat细胞24小时后细胞数目和形态变化。利用Westemblot方法检测硫化砷（0,5,10uM）作用Jurkat细胞24小时后胞内CleavedNotchl蛋白和C．myc蛋白变化情况。结果：@MTT结果提示硫化砷在一定浓度范围内对Jurkat细胞有明显的抑制作用（P〈0．05）,并呈浓度和时间依赖性。②显微镜下观察到硫化砷处理Jurkat细胞24小时后,细胞数目随药物浓度增加而减少,而其细胞碎片随着药物浓度增加而增加。@Westernblot结果显示硫化砷处理24小时后,胞内CleavedNotchl蛋白、C-myc蛋白下调,并随浓度增加下降的更为明显。结论：硫化砷对Jurkat细胞生长具有抑制作用．其机制可能通过影响Notch信号通路起作用。     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

苜蓿素对人直肠癌SW1116细胞增殖和凋亡的作用及其机制

本文探讨苜蓿素对人直肠癌SW1116细胞增殖和凋亡的作用及其机制.采用MTr法检测苜蓿素对SW1116细胞生长的抑制作用;AO/EB染色后观察凋亡细胞形态;流式细胞术检测对细胞周期的影响;DNA片段化分析对细胞凋亡的作用;Westem blot检测对Bcl-2和Bax蛋白表达的影响.结果显示,苜蓿素可明显抑制SW1116细胞的增殖,呈剂量依赖性;AO/EB染色观察到给药组出现细胞凋亡特征;苜蓿素可阻滞细胞于G1/G1期和增加SW1116细胞凋亡率;凝胶成像分析仪检测到典型的DNA阶梯状条带;苜蓿素可呈剂量依赖地减弱Bcl-2和增强Bax蛋白表达.上述结果表明,苜蓿素可显著抑制人直肠癌SW1116细胞的增殖,使细胞阻滞于G1/G1期,并可诱导细胞凋亡,其机制可能与下调Bcl-2和上调Bax蛋白有关.     

鸟氨酸脱羧酶基因反义RNA对淋巴瘤细胞Jurkat生长的影响

探讨鸟氨酸脱羧酶(ornithine decarboxylase,ODC)反义RNA是否对人淋巴瘤细胞Jurkat的生长具有抑制作用。含反义RNA的真核表达载体pcDNA3.1(+)/Rodc用脂质体转染Jurkat细胞,G418筛选ODC表达抑制的细胞株,MTS法分析细胞增殖,Western blotting检测细胞中ODC蛋白表达水平,半定量RT-PCR检测细胞中ODC mRNA含量,流式细胞术检测细胞周期的变化,DNA片段化分析细胞凋亡。结果显示,成功获得ODC表达抑制的淋巴瘤细胞株J/o,ODC反义RNA转染细胞后,引起Jurkat细胞生长缓慢和S/G2细胞周期停滞,细胞对抗癌药物DFMO敏感性显著增加。由此证明,ODC反义RNA能抑制人T淋巴瘤Jurkat细胞的生长,具有治疗人白血病的潜在应用价值。     

活化型高分子量激肽原潜在的抗肿瘤作用及其分子机制

高分子量激肽原是血浆中一种多功能的糖蛋白,与血液凝固的启动、 补体反应及炎症发生等有密切关系.新近的研究显示,活化型高分子量激肽原 具有潜在的抗肿瘤作用.本文综述活化型高分子量激肽原在细胞粘附和血管 生成中发挥的抑制作用及其活性区域,抑制细胞迁移、增殖并诱导细胞凋亡 的作用,及其在细胞表面的作用位点和分子机制.活化型高分子量激肽原作用 机制,包括抑制细胞DNA的从头合成,使细胞周期蛋白D1表达下降,以及通过 影响细胞内信号通路发挥其活性效应等.深入研究活化型高分子量激肽原在 细胞表面作用的信号转导通路可能是今后抗肿瘤研究途径之一.     

小檗碱对急淋白血病的在体抗肿瘤及与Ara-C的联合作用研究

本实验旨在研究小檗碱(berberine, BBR)对急淋白血病细胞Jurkat的在体抗肿瘤作用及与阿糖胞苷(cytosine, Ara-C)的联合作用。本研究通过体外培养Jurkat细胞株,将细胞注射至裸鼠皮下,建立皮下移植瘤模型,待肿瘤体积长至约100 mm~3时,随机分为两组:对照组和小檗碱处理组,分别口服PBS、200 mg/kg小檗碱,隔天给药一次,隔天记录裸鼠体重及肿瘤体积大小。给药30 d后处死动物,剥离肿瘤组织,称量肿瘤大小,绘制肿瘤生长曲线、体重图及瘤重图,计算抑瘤率;采用免疫组化法检测移植瘤组织中细胞核增殖抗原Ki-67的表达水平。同时,本研究用一线用药阿糖胞苷(cytarabine, Ara-C)与小檗碱联合处理Jurkat细胞,研究两药联用的药效。发现小檗碱处理组移植瘤大小受到明显的抑制,瘤块组织中Ki-67的表达明显降低;Ara-C与小檗碱能协同抑制Jurkat细胞的增殖,联合指数(combination index, CI)1。本研究发现小檗碱能够抑制Jurkat细胞皮下移植瘤的增殖,在体内发挥抗急淋白血病活性,并能与一线用药Ara-C协同产生作用。     

棉酚对Jurkat T细胞增殖与凋亡的影响

研究棉酚(gossypol)对Jurkat T细胞增殖和凋亡的影响及可能机制.将不同浓度的棉酚作用于Jurkat T细胞,用MTS比色法检测细胞存活率;以膜联蛋白V-PE染色分析细胞凋亡;用Hoechst33342染色观察核形态:用荧光染料Mitocapture结合流式细胞术和激光共焦显微镜检测线粒体跨膜电位变化.结果显示棉酚作用Jurkat T细胞24 h、48 h、72 h,其IC50值分别为77.2μmol/L、57.3 μmol/L、23.3 μmol/L,对细胞的抑制作用与药物存在时间-剂量依赖关系;终浓度为8、16、32 μmol/L的棉酚处理24 h后的细胞凋亡率分别为5.30%、15.20%、51.19%,对照组凋亡率为3.43%;高浓度棉酚作用后大量细胞核呈现染色质固缩、核碎裂和致密浓染等凋亡特征;随着浓度增加细胞线粒体跨膜电位明显降低.研究表明棉酚能有效抑制Jurkat T细胞增殖和诱导其发生凋亡,并呈现出时间-剂量依赖关系,其诱导凋亡的作用可能依赖于线粒体途径.     

光敏剂HMME对人骨肉瘤细胞U-2OS的光动力作用及其作用机制的初步研究

目的：探讨光敏剂（HMME）介导的光动力疗法对人骨肉瘤细胞U-2OS的杀伤效应及机制研究。方法：使用不同浓度（0、10、20、30、40μg.ml-1）的光敏剂,采用不同光照能量（0、3、6、9 J.cm-2）照射人骨肉瘤细胞,与空白对照组、药物对照组（无光照但加光敏剂）和光照对照组（不加光敏剂但加光照）进行比较,MTT法检测细胞的存活率,选择半数有效量药物浓度和光照能量,作为实验组。以空白对照组为对照,采用Hoechst33342染色法,观察细胞凋亡情况。用western blot方法检测细胞凋亡蛋白caspase-7、caspase-9和PARP-1。结果：MTT结果显示,空白对照组、药物对照组和光照对照组对细胞存活率在96.7%和100%之间,药物的半数有效量为40.1μg.ml-（16 J.cm-2）和25.0μg.ml-（19 J.cm-2）。Hoechst33342染色法观察到实验组细胞明显凋亡。westen blotting检测结果,实验组与对照组相比,caspase-7、caspase-9和PARP-1表达明显增高。结论：HMME-PDT对人骨肉瘤细胞U-2OS有显著的杀伤效应,且呈光敏剂浓度和光照强度依赖性,其杀伤效应与caspase途径相关。     

Prosaposin对细胞增殖和凋亡的调控及其分子机制

为研究鞘脂激活蛋白原(Prosaposin)对细胞增殖、细胞凋亡的调控及其可能的分子机制, 以pcDNA3.1 in NIH3T3阴性对照细胞株和过表达prosaposin的Psap-Myc in NIH3T3细胞株为模型, 噻唑蓝(MTT)比色法检测prosaposin对细胞增殖的影响; Annexin V联合碘化丙啶(Propidium iodide, PI)法检测血清饥饿状态下prosaposin对细胞凋亡的影响; Western blotting检测PI3K/Akt信号通路中蛋白磷酸化水平的变化; Real-time PCR检测PI3K/Akt信号通路下游靶分子表达水平的改变。结果表明prosaposin可活化PI3K/Akt信号通路, 提高Akt^Ser473的磷酸化水平, 抑制细胞周期抑制基因P27^KIP1的表达, 上调细胞周期蛋白Cyclin D1的表达, 促进细胞周期从G1→S期进展; 诱导survival基因cIAP1、cIAP2的表达, 促进细胞存活。这些结果提示, prosaposin对细胞增殖和凋亡的调控可能是通过PI3K/Akt信号通路及其下游靶分子进行的。     

Characterization of ouabain-induced phosphoinositide hydrolysis in brain slices of the neonatal rat

The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570±40% of basal) than in adult animals (287±18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus>cortex>brainstem>cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha₁-adrenergic and glutamate receptors. Also ineffective were the K⁺ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K⁺ and veratrine, but not to that of glutamate. Removal of Na⁺ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [³H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [³H]ouabain binding in brain regions. These results suggest an involvement of Na⁺ pump in the hydrolysis of Ptdlns, possibly related to an effect on Na⁺ and Ca²⁺ homeostasis. The immature rat appear to be an useful model for studying the relationship between Na/K-ATPase and inositol metabolism.     

Characterization of [3H]Ouabain Binding Sites in Human Brain, Platelet, and Erythrocyte

[3H]Ouabain binding was investigated in membranes prepared from human brain, erythrocyte, and platelet. Scatchard analysis of [3H]ouabain binding to human hypothalamic membranes revealed a single class of noninteracting binding sites with an apparent affinity constant (KD) of 21 nM. Though the number of [3H]ouabain binding sites was lower in human platelets than in erythrocytes, both tissues exhibited a single class of high-affinity binding sites with an apparent KD similar to that found in human brain. Specific [3H]ouabain binding in basal ganglia tissue from patients with Huntington's disease was more than 50% lower than in tissue from age- and sex-matched controls. These results, along with previous findings in rat brain, suggest that high-affinity [3H]ouabain binding labels the neuronal form of Na, K-ATPase in human brain, and may prove useful in quantitating this enzyme in postmortem brain samples.     

去甲斑蝥素对白血病Jurkat细胞增殖的影响

目的:观察不同浓度去甲斑蝥素(NCTD)对急性T淋巴细胞白血病细胞株Jurkat增殖的影响。方法:体外培养的Jurkat细胞,用0、5、10、20、40 mg/LNCTD作用6、12、24、48、72 h后,通过倒置显微镜观察细胞形态、密度变化,MTT法检测细胞增殖抑制率,确定其最佳作用浓度及时间。然后将细胞分为NCTD组、长春新碱组、NCTD+长春新碱组,作用24、48、72 h后观察细胞形态、密度变化,MTT法检测细胞增殖抑制率。结果:倒置显微镜显示:随NCTD浓度增加、作用时间延长,细胞形态不规则,胀大、固缩,分布稀疏,大量死亡;MTT法显示:随NCTD浓度增加、作用时间延长,细胞增殖抑制率渐升高,20 mg/L作用72 h时最高(59.24%)。NCTD组与长春新碱组比较差异无统计学意义(P0.05),NCTD+长春新碱组抑制率(77.40%)明显高于NCTD组,差异有统计学意义(P0.05)。结论:NCTD可以浓度和时间依赖性方式抑制Jurkat细胞的增殖,与长春新碱联合后作用增强。     

Mending Fences: Na,K-ATPase signaling via Ca2+ in the maintenance of epithelium integrity

Na,K-ATPase is a ubiquitous multifunctional protein that acts both as an ion pump and as a signal transducer. The signaling function is activated by ouabain in non-toxic concentrations. In epithelial cells the ouabain-bound Na,K-ATPase connects with the inositol 1,4,5-trisphosphate receptor via a short linear motif to activate low frequency Ca2+ oscillations. Within a couple of minutes this ouabain mediated signal has resulted in phosphorylation or dephosphorylation of 2580 phospho-sites. Proteins that control cell proliferation and cell adhesion and calmodulin regulated proteins are enriched among the ouabain phosphor-regulated proteins. The inositol 1,4,5-trisphosphate receptor and the stromal interaction molecule, which are both essential for the initiation of Ca2+ oscillations, belong to the ouabain phosphor-regulated proteins. Downstream effects of the ouabain-evoked Ca2+ signal in epithelial cells include interference with the intrinsic mitochondrial apoptotic process and stimulation of embryonic growth processes.The dual function of Na,K-ATPase as an ion pump and a signal transducer is now well established and evaluation of the physiological and pathophysiological consequences of this universal signal emerges as an urgent topic for future studies.     

苦参碱对哇巴因诱导钠电流改变的调节作用

目的:探讨苦参碱拮抗哇巴因诱导的心律失常的作用机制。方法:应用全细胞膜片钳技术记录哇巴因对单个豚鼠心室肌细胞的Na+电流和动作电位时程作用后,观察苦参碱对哇巴因诱导Na+电流和动作电位时程改变的恢复作用。结果:1 5μmol·L-1哇巴因延长APD50从给药前476±40.7 ms增加到744±62.9 ms(n=6,P0.05),APD90从给药前499±84.9 ms增加到775±87.7 ms(n=6,P0.01),100μmol·L-1苦参碱恢复APD50至603±79.0 ms(n=6,P0.05),APD90至630±81.6 ms(n=6,P0.05);2 5μmol·L-1哇巴因可增加钠电流的峰值,在-20 m V电压条件下,5μmol·L-1哇巴因增加INa,由正常-40.9±2.32 p A/p F增加到-55.2±2.26 p A/p F(n=8,P0.05),100μmol·L-1苦参碱减少INa至-34.6±2.14 p A/p F(n=8,P0.05);5μmol·L-1哇巴因右移钠通道的激活曲线,并左移钠通道的失活曲线从而改变通道动力学特性;100μmol·L-1苦参碱可抑制哇巴因诱导的INa的增加,并恢复Na+通道动力学特性接近正常。结论:苦参碱拮抗哇巴因诱导的心律失常机制与其抑制哇巴因诱发细胞水平Na+电流的增加,缩短哇巴因诱发APD的延长有关。     

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.     

白藜芦醇对人子宫内膜癌细胞系AN3CA增殖和凋亡效应及其机制探讨

目的：探讨白藜芦醇（resveratrol,Res）对人子宫内膜癌细胞AN3CA的增殖抑制和凋亡诱导效应及可能存在的机制。方法：应用噻唑蓝（MTT）法检测Res对AN3CA的增殖影响;流式细胞术检测Res对细胞周期分布和凋亡影响：荧光实时定量PCR检测Res对细胞Bcl-2、Bax和MMP-9mRNA表达水平的影响;WesternBlot方法检测Res对PCNA、Bcl-2、Bax及ERK1／2、P—ERK1／2蛋白表达水平的影响。结果：Res对子宫内膜癌细胞AN3CA具有显著的生长抑制作用（P〈0．01）,呈时间-剂量依赖性;不同浓度Res处理细胞G0／G1期比例显著增加伴随S期细胞数的减少;细胞凋亡率明显增高,200Dmol／lRes处理48h凋亡率可达30．96％±2．041％（P〈0．01）。与对照组相比,Res能抑制PCNA的蛋白表达量,增加Bax和降低Bcl-2转录和蛋白水平的表达量。Res在短时间内（0．5—1h）激活ERK1／2的磷酸化表达但随着作用时间延长（4—48h）其表现为抑制效应。结论：Res具有抑制AN3CA细胞增殖,诱导细胞G0／G1期阻滞和凋亡的效应。Res诱导凋亡可能是通过上调Bax,下调Bcl-2发挥作用,其抗癌作用机制可能与ERK1／2通路失调相关。     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

白藜芦醇对人子宫内膜癌细胞系AN3CA 增殖和凋亡效应 及其机制探讨

目的:探讨白藜芦醇(resveratrol,Res)对人子宫内膜癌细胞AN3CA的增殖抑制和凋亡诱导效应及可能存在的机制。方法:应用噻唑蓝(MTT)法检测Res对AN3CA的增殖影响;流式细胞术检测Res对细胞周期分布和凋亡影响;荧光实时定量PCR检测Res对细胞Bcl-2、Bax和MMP-9mRNA表达水平的影响;Western Blot方法检测Res对PCNA、Bcl-2、Bax及ERK1/2、p-ERK1/2蛋白表达水平的影响。结果:Res对子宫内膜癌细胞AN3CA具有显著的生长抑制作用(P<0.01),呈时间-剂量依赖性;不同浓度Res处理细胞G0/G1期比例显著增加伴随S期细胞数的减少;细胞凋亡率明显增高,200μmol/l Res处理48h凋亡率可达30.96%±2.041%(P<0.01)。与对照组相比,Res能抑制PCNA的蛋白表达量,增加Bax和降低Bcl-2转录和蛋白水平的表达量。Res在短时间内(0.5-1h)激活ERK1/2的磷酸化表达但随着作用时间延长(4-48h)其表现为抑制效应。结论:Res具有抑制AN3CA细胞增殖,诱导细胞G0/G1期阻滞和凋亡的效应。Res诱导凋亡可能是通过上调Bax,下调Bcl-2发挥作用,其抗癌作用机制可能与ERK1/2通路失调相关。