胞外ATP影响ZnCl_2诱导烟草悬浮细胞活性和呼吸速率的降低

以烟草悬浮细胞BY-2为材料,探讨细胞外ATP对ZnCl_2诱导的细胞活性降低和呼吸抑制的调节作用.结果表明,随着ZnCl_2浓度逐渐增加,细胞活性、胞外ATP浓度和细胞呼吸速率均随之降低.在400 mmol/L ZnCl_2胁迫细胞的条件下,研究了细胞外ATP对ZnCl_2诱导的细胞活性降低和呼吸抑制的调节作用.结果发现,与400 mmol/L ZnCl_2胁迫下的细胞相比,加入20μmol/L外源ATP到ZnCl2胁迫下的细胞中,会使细胞的活性、胞外ATP浓度和细胞呼吸速率均有显著升高.胞外ATP浓度的变化可能是造成ZnCl_2胁迫下细胞活性受抑制的原因之一,同时胞外ATP对ZnCl_2胁迫下细胞的活性具有一定的调节作用.     

细胞外ATP调节了NaCl胁迫诱导的烟草悬浮细胞的死亡和呼吸的抑制

本文以烟草悬浮细胞BY-2为材料,探讨了胞外ATP对NaCl诱导的细胞死亡和呼吸抑制的调节作用。实验表明,随着NaCl浓度的逐渐上升(50、100、200、400 mmol/L),细胞的死亡水平逐渐上升,而胞外ATP含量和细胞呼吸速率则随NaCl浓度的上升而逐渐下降。本文在200 mmol/L NaCl处理下的细胞中探索了胞外ATP对NaCl诱导的细胞死亡和呼吸抑制的调节作用。结果发现,较之200 mmol/L NaCl胁迫下的细胞,对NaCl胁迫的细胞加入外源ATP(20 μmol/L )其使得细胞死亡水平显著性降低,也使得胞外ATP含量和细胞呼吸速率均有所回升。上述实验观察表明,NaCl胁迫诱导的植物细胞的死亡和呼吸抑制可能和细胞外ATP水平的变化有关,而胞外ATP对NaCl诱导的细胞死亡具有一定的调节作用。     

H+对非洲爪蟾卵母细胞ATP-激活电流的调制作用

研究H^＋对非洲爪蟾卵母细胞内源性ATP受体的调制作用。采用双极电压钳技术记录卵母细胞膜ATP受体介导的电流。实验检测的细胞全部对ATP敏感，应用pH为7．4的ATP（10^-3mol／L）后引起一特征性电流，呈浓度依赖性。改用pH为6．5，6．0，5．5的ATP（10^-3mol／L）后，电流幅值明显减弱。结果表明，pH值降低，可抑制ATP诱导的卵母细胞膜电流。     

氨脉冲酸化蟾蜍膀胱膜上皮细胞的研究

用pH荧光探剂2′，7′-二环（2-羧乙基）-5（6）-羧基荧光黄（BCECF）研究氨脉冲对蟾蜍膀胱上皮细胞的酸化效果，测量细胞内pH（pHi）值的变化。当用25mmol NH4Cl溶液作用于膀胱膜浆液面（S面）时，pHi值负增长0.15，细胞被酸化；而作用于粘液面（M面）时不能酸化细胞，将50цmolBa^2 加到S面，细胞的酸化被阻止。这说明NH4^ 通过上皮细胞底侧膜的K^ 通道进入细胞使pHi降低。用季胺离子（TMA^ ）代替Na^ ，能加大酸化幅度。1mmol amiloride能使pHi回升速度和幅度都大大降低，这表明位于上皮细胞顶膜的Na^ /H^ 交换参与了pHi调节。     

细胞外ATP调节NaCl胁迫诱导的烟草悬浮细胞死亡和呼吸抑制

本文以烟草悬浮细胞BY-2为材料,探讨了胞外ATP对NaCl诱导的细胞死亡和呼吸抑制的调节作用.实验表明,随着NaCl浓度的逐渐上升(50、100、200、400mmol/L),细胞的死亡水平逐渐上升,而胞外ATP含量和细胞呼吸速率则随NaCl浓度的上升而逐渐下降.本文在200mmol/L NaCl处理下的细胞中探索了胞外ATP对NaCl诱导的细胞死亡和呼吸抑制的调节作用.结果发现,较之200mmol/L NaCl胁迫下的细胞,对NaCl胁迫的细胞加入外源ATP(20μmol/L)其使得细胞死亡水平显著性降低,也使得胞外ATP含量和细胞呼吸速率均有所回升.上述实验观察表明,NaCl胁迫诱导的植物细胞的死亡和呼吸抑制可能和细胞外ATP水平的变化有关,并且胞外ATP对NaCl诱导的细胞死亡具有一定的调节作用.     

MG-132诱导宫颈癌细胞表达HPV病毒蛋白E6和E7

使用不同浓度的蛋白酶体抑制剂MG-132处理官颈癌细胞系CaSki和HeLa S3细胞,WesternBlot检测发现2-5 mg/L MG-132 37℃处理3 h明显增强CaSki细胞中人类乳头瘤病毒HPV E6和E7蛋白的表达量,增加MG-132浓度或延长处理时间并未见E6和E7蛋白量的增加.用相同的方法处理HeLa s3细胞,也观察到HPV E7蛋白的增加.XTT活细胞分析和台盼蓝死细胞检测显示2 mg/L MG-132处理癌细胞3 h,其细胞死亡率10%.这一发现将有可能协助提高以E6、E7蛋白作靶位点的生物导弹疗法的效果.     

细胞外ATP影响铜诱导的细胞死亡和H_2O_2的产生（英文）

细胞外ATP是植物体中许多生理过程的调节信号.胞外ATP可以影响铜离子诱导的细胞死亡和H_2O_2的产生.100~700/μmol/L CuCl_2导致烟草悬浮细胞的死亡量显著上升,而且胞内H_2O_2和胞外H_2O_2的含量也随之上升.选择了300/μmol/L CuCl_2研究铜离子导致的细胞死亡量上升和H_2O_2产生的过程.结果表明,此浓度的CuCl_2提升了NADPH氧化酶的活性,而加入DPI(NADPH氧化酶抑制剂)缓解了CuCl_2引起的细胞死亡和H_2O_2的产生,这说明CuCl_2引起细胞死亡和H_2O_2产生与NADPH氧化酶活性的增加相关.加入50μmol/L ATP进一步增加了CuCl_2引起的细胞死亡、H_2O_2的产生、NADPH氧化酶.而DPI预处理后,外源ATP并未引起变化.这一实验表明,胞外ATP可以通过刺激NADPH氧化酶影响铜离子引起的细胞活性和H_2O_2产生的变化.     

ATP对胞内钙信号的调控机制研究

以大鼠胰腺β细胞和INS-1β细胞系为研究对象,采用显微荧光测钙技术,研究了胞外ATP对胞内Ca^2＋信号的影响,初步探讨了其作用机制．实验表明：胞外ATP能够分别使大鼠胰腺β细胞和INS-1细胞内的游离Ca^2＋浓度显著升高,但2种细胞的钙信号来源不同．在大鼠胰腺β细胞中,胞外ATP主要通过动员胞内钙库释放而引起胞浆内Ca^2＋浓度显著增高;而在INS-1细胞内,胞外ATP主要通过引起胞外Ca^2＋内流而引起胞浆内Ca^2＋浓度增加．     

新型tacrine双联体对培养大鼠海马神经元NMDA受体作用位点的研究

目的：研究新型tacrine双联体bis（7）-tacrine对NMDA受体的作用位点.方法：大鼠海马神经元原代培养,全细胞膜片钳记录培养大鼠海马神经元上NMDA激活电流变化.结果：细胞外液pH值从8.1改变到6.7,在细胞外液中加入二硫苏糖醇（2 mmol/L）、精胺（10 mol/L）、镁离子（50～500 mol...     

ATP刺激巨噬细胞[Ca2+]i升高和氧自由基增加及大黄酸的抑制作用研究

研究了ATP刺激的大鼠腹腔巨噬细胞[Ca2 ]i升高与氧自由基(ROS)产生的关系和大黄酸抑制作用特征.提取大鼠腹腔巨噬细胞,利用Ca2 探针Fura-2检测单细胞胞内自由Ca2 浓度([Ca2 ]i)变化,同时利用NBT还原反应强度检测同一细胞ROS产生能力.结果发现无ATP刺激的巨噬细胞的ROS产生量较低;1mmol/L ATP刺激巨噬细胞单细胞后诱发[Ca2 ]i显著升高由胞内Ca2 释放和胞外Ca2 内流组成,同时ROS产生增强2倍;胞外无Ca2 条件下ROS产生随着[Ca2 ]i下降而减少.10-5和10-4mol/L浓度大黄酸对1mmol/L ATP刺激巨噬细胞单细胞的[Ca2 ]i升高和ROS产生有剂量依赖性的抑制作用;多细胞的统计分析表明10-5和10-4mol/L浓度大黄酸分别抑制了[Ca2 ]i峰值的49%和84%,同时抑制了ROS产生的59%和81%.因此认为ATP刺激大鼠腹腔巨噬细胞诱发的[Ca2 ]i升高介导了ROS的产生,大黄酸剂量依赖性的抑制ATP刺激的细胞[Ca2 ]i升高和ROS产生能力,并提示大黄酸抑制细胞[Ca2 ]i升高是其抑制ROS产生的重要机制.     

不同诱导条件对树鼩骨髓间充质干细胞向成骨细胞分化的影响

目的 探讨不同诱导条件对树鼩骨髓间充质干细胞（BM-MSCs）体外向成骨细胞分化的影响。方法 取 P3代树鼩骨髓间充质干细胞分成7组进行诱导。A组：高糖DMEM+1μmol/L地塞米松+100μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠;B组：高糖 DMEM+50 ng/mL BMP-2;C组：高糖 DMEM+80 ng/mL BMP-2;D组：高糖DMEM+50μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠+20ng/m L BMP-2;E组：高糖 DMEM+1μmol/L地塞米松 +100μmol/L维生素C+20 mmol/Lβ-磷酸甘油钠;F组：高糖DMEM+100μmol/L地塞米松+100μmol/L维生素 C+10 mmol/Lβ-磷酸甘油钠;G组：DMEM/F12+0.1μmol/L地塞米松 +50μmol/L维生素C+10 mmol/Lβ-磷酸甘油钠。诱导18 d后进行碱性磷酸酶和茜素红染色鉴定其诱导分化情况。结果 每一组碱性磷酸酶染色呈不同程度的阳性,茜素红染色可在A、B、C、D和E组观察到明显矿化结节。结论 A组、B组、C组、D组和 E组可以诱导树鼩BM-MSCs向成骨细胞分化,其中以A组和B组效果最为突出。     

Digital image processing of intracellular pH in gastric oxyntic and chief cells

Cytosolic pH (pHi) is a critically regulated determinant of intracellular function. Several mechanisms for pHi regulation in different tissues have been found, such as direct proton pumping, Na/H exchange, Cl/HCO3 exchange, NaHCO3 cotransport, and Na/H/Cl/HCO3 obligatorily linked. All these studies have used either single cells or cell populations assumed to be behaving homogeneously. Most tissues consist of more than one cell type, so it would be desirable to examine pHi regulation simultaneously in many identified individual cells, particularly in epithelia where disaggregation and purification of isolated cells destroys the normal distinction between luminal and serosal environments. We have used a pH-sensitive fluorescent dye, BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein) and digital image processing to study pHi regulation simultaneously in the oxyntic cells (OC) and chief cells (CC) of gastric glands isolated from rabbit stomach. CCs become markedly more acidic upon removal of external Na (Nao), but pHi is restored rapidly on return to normal Nao, with or without Cl. Oxyntic cell pHi is much less affected by Nao. Conversely, OCs become strongly more alkaline on removal of external Cl (Clo), pHi being restored when Clo is replaced with or without Na, whereas CCs are relatively insensitive to Clo. Therefore, Na/H exchange is dominant over Cl/HCO3 exchange in CCs, but in the neighbouring OCs, Cl/HCO3 outweighs the Na/H mechanism, a heterogeneity that correlates with the functions of the two cell types.     

Chaetoglobosins E诱导乳腺癌MCF-7细胞凋亡作用机制研究

为探究Chaetoglobosins E(ChE)对肿瘤细胞增殖和凋亡的影响,体外培养人乳腺癌MCF-7细胞、人膀胱癌T-24细胞、人黑色素瘤C8161细胞、人白血病U937细胞,用不同浓度的ChE分别作用于4种细胞24 h或48 h,MTT法检测4种肿瘤细胞的增殖情况;为进一步研究其作用机制,Hoechst 3334...     

Modulation of ATP-sensitive K+ channels in skeletal muscle by intracellular protons

Since their discovery in cardiac muscle, ATP-sensitive K+(KATP) channels have been identified in pancreatic beta-cells, skeletal muscle, smooth muscle and central neurons. The activity of KATP channels is inhibited by the presence of cytosolic ATP. Their wide distribution indicates that they could have important physiological roles that may vary between tissues. In muscle cells the role of K+ channels is to control membrane excitability and the duration of the action potential. In anoxic cardiac ventricular muscle KATP channels are believed to be responsible for shortening the action potential, and it has been proposed that a fall in ATP concentration during metabolic exhaustion increases the activity of KATP channels in skeletal muscle, which may reduce excitability. But the intracellular concentration of ATP in muscle is buffered by creatine phosphate to 5-10 mM, and changes little, even during sustained activity. This concentration is much higher than the intracellular ATP concentration required to half block the KATP-channel current in either cardiac muscle (0.1 mM) or skeletal muscle (0.14 mM), indicating that the open-state probability of KATP channels is normally very low in intact muscle. So it is likely that some additional means of regulating the activity of KATP channels exists, such as the binding of nucleotides other than ATP. Here I present evidence that a decrease in intracellular pH (pHi) markedly reduces the inhibitory effect of ATP on these channels in excised patches from frog skeletal muscle. Because sustained muscular activity can decrease pHi by almost 1 unit in the range at which KATP channels are most sensitive to pHi, it is likely that the activity of these channels in skeletal muscle is regulated by intracellular protons under physiological conditions.     

活性氧与NO在SO2诱导蚕豆气孔运动中的作用

以蚕豆叶表皮为材料,研究SO2胁迫时叶面气孔运动及其调节途径.研究发现,用浓度1～200μmol/L的SO2衍生物(亚硫酸钠与亚硫酸氢钠混合液)处理蚕豆叶下表皮后,气孔开度明显减小,气孔保卫细胞内活性氧(ROS)、一氧化氮(NO)和钙离子(Ca2+)水平显著升高.采用抗氧化剂抗坏血酸和过氧化氢酶,钙离子干扰剂EGTA和LaCl3,以及NO合成抑制剂NaN3与NO清除剂c-PTIO,分别与SO2衍生物同时作用时,SO2诱发的气孔关闭效应得到有效缓解,保卫细胞内ROS、NO和Ca2+水平随之改变.抗氧化剂和NO干扰剂能阻止SO2诱导的胞内ROS、NO和Ca2+水平升高;EGTA和LaCl3能降低SO2诱导的胞内NO和Ca2+升高,但不影响ROS水平.研究结果表明,较高浓度SO2能诱导气孔关闭,SO2胁迫诱导ROS和NO合成增加,ROS和NO通过钙信号系统调节气孔开度.     

快速诱导细胞凋亡方法探讨

[目的]利用流式细胞仪检测细胞凋亡在基础研究、疾病诊断及预后预测及评价中具有重要的应用价值.补偿调节所需的单阳染色管的制备,是流式细胞术检测凋亡过程的一个重要步骤.探讨了快速诱导不同细胞系和原代细胞凋亡的方法,优化用于检测细胞凋亡的流式单阳染色管快速制备.[方法](1)分别用100 μmol/L H2O2、体积分数4％...     

α-生育酚琥珀酸酯对乳腺癌细胞中IAPs家族蛋白表达的影响

目的检测α-生育酚琥珀酸酯(-αTOS)对MCF7和MDA-MB-453乳腺癌细胞增殖以及细胞中凋亡抑制蛋白家族(IAPs)表达的影响。方法采用四甲基偶氮唑蓝(MTT)法测定-αTOS对人乳腺癌细胞MCF7和MDA-MB-453增殖的抑制作用,细胞分别接种于96孔板后用浓度为51、0、255、0、75、1001、25和150μmol/L的-αTOS处理48 h后检测;细胞经50μmol/L、100μmol/L-αTOS处理122、4、48 h后,用RT-PCR的方法检测细胞内c-IAP1和c-IAP2 mRNA的转录水平;用Western Blot的方法检测c-IAP1和c-IAP2的蛋白质表达水平。结果-αTOS对人乳腺癌细胞MCF7和MDA-MB-453有显著的增殖抑制作用,并表现为剂量依赖性,-αTOS对MCF7和MDA-MB-453的IC50值(细胞半数死亡的药物浓度)分别为132μmol/L和74μmol/L;RT-PCR结果显示,-αTOS使两个细胞系的c-IAP1的转录水平显著下降,且呈时间依赖性;但对c-IAP2的转录水平没有明显影响;Western Blot的结果发现-αTOS使得这两种细胞系的c-IAP1蛋白质翻译水平也呈下降趋势,该结果与mRNA转录水平下降相一致,对c-IAP2蛋白质的表达几乎无影响。因此,α-TOS可能通过抑制c-IAP1蛋白的表达而抑制MCF7和MDA-MB-453乳腺癌细胞的生长增值,a-TOS有望开发成为新的预防和治疗乳腺癌的有效药物。     

Arginine vasopressin enhances pHi regulation in the presence of HCO3- by stimulating three acid-base transport systems

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.     

Effects of hydrogen peroxide on in vitro infection of Cryptosporidium parvum

The effect of hydrogen peroxide (H2O2) on Cryptosporidium parvum infection and the protective function of free radical scavengers were studied in cultured bovine fallopian tubes epithelial cells. H2O2 treatment at 500 μmol/L and 1 mmol/L significantly inhibited excystation of bleach-treated oocysts (p ＜ 0. O1 ). At 48 h postinoculation,H2O2 at 500 and 750 μmol/L resulted in a significant (p ＜ 0.01) decrease of C. parvum infection by 35.77% and 58.16% respectively in the cultured cells, when compared with the untreated control. Surprisingly, the infection was significantly increased from 22.21% to 39.33 % ( p ＜ 0.05) with lower concentration of H2 O2 (50 ～ 200 μmol/L). The inhibitory and stimulatory effects of H2O2 treatment on C. parvum infection were, to a certain extent, abolished in the presence of reduced glutathione or mannitol. These observations indicate that reactive oxygen species, such as H2O2, may play an important role in C. parvum infection.     

pH-sensitive activation of the intracellular-pH regulation system in squid axons by ATP-gamma-S

The regulation of intracellular pH (pHi) is essential for normal cell function, and controlled changes in pHi may play a central role in cell activation. Sodium-dependent Cl-HCO3 exchange is the dominant mechanism of pHi regulation in the invertebrate cells examined, and also occurs in mammalian cells. The transporter extrudes acid from the cell by exchanging extracellular Na+ and HCO3- (ref. 9) (or a related species) for intracellular Cl- (refs 3, 4). It is blocked by the stilbene derivatives DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate, ref. 10) and SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonate, ref. 3), and has a stoichiometry of two intracellular H+ neutralized for each Na+ taken up and each Cl- extruded by the axon. Because the inwardly-directed Na+ concentration gradient is sufficiently large to energize both the HCO3- influx and Cl- efflux, this electroneutral exchanger could be a classic secondary active transporter, thermodynamically independent of ATP hydrolysis. However, at least in the squid axon, the exchanger has an absolute requirement for ATP (ref. 3). Thus, a major unresolved issue is whether this Na-dependent Cl-HCO3 exchanger stoichiometrically hydrolyses ATP (the pump hypothesis), or whether ATP activates the transporter by a mechanism such as phosphorylation or simple binding (the activation hypothesis). We have now explored the role of ATP in pHi regulation by dialysing axons with the ATP analogue ATP-gamma-S. In many systems, ATP-gamma-S is an acceptable substrate for protein kinases, whereas the resulting thiophosphorylated proteins are not as readily hydrolysed by phosphatases as are phosphorylated proteins. Our results rule out the pump hypothesis, and show that the basis of the axon's ATP requirement is the pH-dependent activation (by, for instance, phosphorylation or ATP binding) of the exchanger itself, or of an essential activator.