线粒体PT孔参与甘草诱导MGC-803细胞凋亡的调控

不久前我们从中药中首次筛选发现了甘草能显著诱导胃癌MGC-803细胞凋亡,本文进一步研究甘草诱导MGC-803细胞凋亡过程中凋亡百分率、线粒体膜电位、胞内游离钙、DNA电泳和细胞膜通透性以及染色质DNA凝聚的时相变化,并研究了线粒体PT孔专一抑制剂环孢菌素A(CsA)对凋亡过程的影响.我们观察到,细胞膜通透性增强、胞内游离钙升高和线粒体膜电位下降为细胞凋亡的早期事件,先于凋亡峰出现、染色质凝聚和DNA电泳梯状条带出现,CsA明显抑制线粒体膜电位下降,细胞膜通透性增强和胞内游离钙变化,并极大程度地延迟细胞凋亡过程.结果提示,钙和CsA敏感性的线粒体PT孔开放参与甘草提取物诱导MGC-803细胞凋亡的调控.     

球孢菌素A和Bcl—2高表达对EGTA诱导HL—60细胞凋亡的影响

研究线粒体PT孔专一抑制剂环孢菌素A（CsA）和Bcl-2高表达对EGTA诱导HL－60细胞凋亡的影响。流式细胞仪检测凋亡峰、染色质凝聚的PI和Hoechst33342荧光双染观察、DNA梯状条带分析均表明,CsA明显促进EGTA诱导的HL－60细胞凋亡,而Bcl-2高表达完全阻断细胞凋亡的发生,借助荧光探针rhodamine123和CMXRos研究细胞凋亡过程线粒体△ψm下降,而Bcl-2高表达使HL－60细胞的线粒体△ψm提高了近1倍,并完全抑制EGTA诱导的线粒体△ψm下降。     

流式细胞术研究细胞凋亡的方法与技术

细胞发生凋亡时,会伴随着一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质Ca2+浓度升高,DNA片段化及含量变化等特点.应用流式细胞术进行细胞凋亡的研究,对于探讨胚胎发育、衰老以及研究肿瘤的发生、发展和转化等病理生理过程和病毒感染及免疫等具有十分重要的意义.本文就细胞凋亡的特征、基于细胞膜功能的流式细胞术检测方法和基于细胞器功能的流式细胞术检测方法等关键性问题进行了阐述.     

三尖杉酯碱诱导的HL-60细胞凋亡的钙调节

三尖杉酯碱 (harringtonine ,HT)是一种对急性粒细胞白血病、急性单核细胞白血病有良好疗效的抗癌药物 ,可在很宽的剂量范围内迅速诱导HL -6 0细胞凋亡 .细胞外钙离子螯合剂EGTA不抑制抗癌药物HT、喜树碱 (campothecin ,CAM )诱导的-细胞凋亡 ;而细胞内Ca ²⁺螯合剂BAPTA AM却可抑制该过程 .与此相一致 ,HT和CAM也不能诱导胞内Ca ²⁺已排空的HL -6 0细胞凋亡 ,说明HT ,CAM诱导的HL- 6 0细胞凋亡依赖于胞内Ca ²⁺但是HT ,CAM诱导HL -6 0细胞凋亡过程中胞内自由Ca ²⁺浓度变化不大 .利用视频反差增强显微术 (videoenhance mentcontrastmicroscopy ,VEC)研究了单个HL- 6 0细胞凋亡过程中胞内Ca ²⁺分布的动态变化 ,结果表明HT诱导HL -6 0细胞凋亡过程存在胞内Ca ²⁺由胞质向核的位移 .     

脉冲电场对粒细胞内Ca2+浓度影响的动态变化

利用激光共聚焦扫描显微镜和装有CCD系统的荧光显微镜,研究在单脉冲电场作用下经fluo-3/AM标记的鸡胚小脑粒细胞内自由Ca2+浓度(i)的动态变化过程.结果表明:在单个电脉冲作用下,细胞内Ca2+浓度立刻升高并达到其最大值.Ca2+浓度升高的幅度以及升高的速率具有电场强度的依赖性.当细胞外Ca2+被过量的EGTA络合或细胞膜上的Ca2+通道被La3+堵塞后,细胞内的Ca2+浓度仍然升高.细胞内不同区域的Ca2+浓度同时升高;两极内的Ca2+浓度早于胞体的Ca2+浓度达到最大值并迅速恢复.     

线粒体Ca~(2 )转运与细胞代谢调节

线粒体具有一套完整的Ca2 转运系统 ,包括两条摄取途径和三条释放途径。生理条件下 ,它们在细胞胞质与线粒体钙稳态维持以及细胞能量代谢中起重要作用 ,线粒体从胞质摄取的Ca2 可激活某些Ca2 敏感的呼吸酶和代谢过程。病理条件下 ,线粒体Ca2 转运发生紊乱 ,通过线粒体通透性转换导致细胞坏死或凋亡     

胸腺细胞和细胞器水平的线粒体膜电势研究

以地塞米松(DEX)诱导小鼠胸腺细胞凋亡;利用PI和AnneXin V/PI流式细胞术分别检测细胞晚期和早期凋亡;利用JC-1和DiOC_6(3)/PI在细胞水平检测凋亡中线粒体膜电势(△ψm)变化：抽提线粒体,利用JC-1直接染色技术检测现存线粒体△ψm情况。实验结果显示,DEX显著诱导胸腺细胞早期和晚期凋亡,凋亡细胞主要来自G_0/G_1期;细胞水平可见DEX介导与△ψm相关的J-aggregate和DiOC_6(3)可染性降低,同时介导线粒体数量显著降低,6h细胞膜完整性无显著变化：单纯线粒体检测结果显示,多数线粒体维持正常△ψm。提示,DEX介导胸腺细胞凋亡中线粒体数量降低,现存线粒体多保持着正常△ψm以维持凋亡过程细胞能量供给。     

外源钙调素对粟酒裂殖酵母细胞增殖的影响

外源钙调素（CaM）对粟酒裂殖酵母（Schizosaccharomycespombe）细胞增殖的影响。实验结果表明外源CaM能明显抑制粟酒裂殖酵母细胞的增殖,其作用方式是延长了粟酒裂殖酵母细胞生长的延滞期。抗粟酒裂殖酵母CaM抗体、TFP及Phenyl-SepharoseCL－4B能降低CaM对细胞生长的抑制作用,而Ca2+及Ca2+螫合剂EGTA对CaM的抑制作用均无影响。以上结果提示,外源CaM对粟酒裂殖酵母细胞增殖的抑制作用可能是由于胞外CaM激活了细胞膜上的Ca2+泵,使胞内Ca2+浓度降低所致。     

Zn~(2+)对PTP开放和细胞色素c释放的浓度依赖性双向调节

研究Zn2+对Ca2+介导线粒体通透过渡孔道(PTP)开放和线粒体细胞色素c释放的影响,及其与线粒体膜电位(ΔΨm)和Ca2+介导的线粒体Ca2+释放(mCICR)之间的关系.提取大鼠肝线粒体,通过紫外分光光度仪检测不同浓度Zn2+作用下Ca2+介导的PTP开放状态;采用荧光分光光度仪测定不同浓度Zn2+作用下线粒体膜电位的变化;采用双波长双光束紫外分光光度仪检测不同浓度Zn2+作用下测试体系内Ca2+浓度的变化,以反映线粒体Ca2+的转运情况(即mCICR);通过免疫印迹法检测不同浓度Zn2+作用下Ca2+介导的线粒体细胞色素c的释放.高浓度Zn2+完全抑制Ca2+介导的PTP开放和细胞色素c释放.一定浓度的Zn2+部分抑制Ca2+介导的PTP开放和细胞色素c释放.适当浓度Zn2+自身介导PTP开放和细胞色素c释放.低浓度Zn2+加速Ca2+介导PTP开放和Ca2+释放;高浓度和一定浓度Zn2+分别完全或部分破坏ΔΨm;高浓度Zn2+完全抑制mCICR.当抑制mCICR时,Ca2+和Zn2+对PTP开放和细胞色素c释放的作用完全抑制.结果表明,Zn2+以浓度依赖方式双向调节PTP开放和细胞色素c释放.Zn2+的作用可能与Zn2+破坏ΔΨm和影响mCICR相关.     

甘草水溶物诱导MGC-803细胞凋亡中胞内Ca2+、pH与线粒体△ψm的变化

细胞凋亡时发生染色质凝聚、DNA呈梯状片段化、胞体皱缩、质膜起泡、形成凋亡小体等典型的形态和生化变化,并受凋亡信号系统激活所导致的细胞内一系列酶的活性以及凋亡相关基因表达的变化调控.近年来的研究表明,细胞内Ca2 、pH、线粒体膜电位(△Ψm)等生物物理性状的改变与细胞凋亡信号系统的调控有密切的关系,甚至是决定性的因素.糖皮质激素能非常有效地诱导胸腺细胞发生凋亡,已证实在这典型的凋亡过程中,细胞内Ca2 浓度变化是至为重要的环节,处理细胞早期就可见到细胞内Ca2 的持续升高,然后是DNA片段化.尽管也有文献报道在无Ca2 基质…     

线粒体PT孔参与甘草诱导MGC—803细胞凋亡的调控

In our previous studies, we have discovered that the extract of glycyrrhiza uralensis Fisch (EGUF) can induce obvious apoptosis in gastric cancer cell Line MGC-803. Here, further investigation was carried on about the time-lapse changes of mitochondria transmembrane potential, intracellular free calcium ions, DNA electrophoresis, plasma membrane permeability and chromatin condensation during the apoptotic process of MGC-803 induced by EGUF and the influences of MPT-specific inhibitor Cyclosporin A(CsA) on these changes. Enhancement of plasma membrane permeability with PI staining, increase of intracellular free calcium ion and decrease of mitochondria transmembrane potential are early events in apoptotic cascades, prior to the appearances of apoptotic peak, chromatin condensation and DNA ladder. CsA significantly inhibited enhancement of plasma membrane permeability, change of intracellular free calcium ions and decrease of mitochondria transmembrane potential, also greatly delayed the progress of apoptosis. Thus, our results suggest that calcium and CsA-sensitive MPT is involved in the apoptosis of MGC-803 induced by EGUF.     

外源Ca2+对高温强光胁迫下滨梅幼苗的保护效应

以10 mmol/L CaCl2溶液处理滨梅幼苗叶片后,置于培养箱于(40±2)℃高温、光照强度(1 200±50)μmol·m-2·s-1下培养,定期测定有关生理生化指标,以探讨外源Ca2+对高温强光胁迫下滨梅幼苗的保护效应.结果显示:(1)与蒸馏水处理组相比,Ca2+处理使高温强光胁迫下滨梅幼苗叶片的脯氨酸含量显著升高,可溶性糖含量变化不明显,根系活力小幅降低;Ca2+处理有效抑制了高温强光下膜透性的加大,提高和保护了Ca2+-ATPase的活性.(2)采用Ca2+螯合剂EGTA或钙调素拮抗剂TFP对滨梅幼苗叶片同法处理并同条件胁迫时,与Ca2+处理相比,滨梅幼苗的脯氨酸、可溶性糖含量、Ca2+-ATPase活性和根系活力均明显下降,膜透性加大.研究表明,Ca2+处理能提高滨梅幼苗对高温强光的耐受性;Ca2+信号系统参与了胁迫过程中的渗透物质和Ca2+-ATPase活性等的调节.     

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.     

Modulation of the matrix redox signaling by mitochondrial Ca~(2+)

Mitochondria sense,shape and integrate signals,and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance,the molecular nature of the proteins involvedin mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes energy metabolism through the activation of matrix dehydrogenases and downstream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)+ ratio,but at the same time will increase reactive oxygen species(ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state,which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redoxsensitive sensors,real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca2+ combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca2+ and redox signals and their impact on cell function. In this review,we describe mitochondrial Ca2+ handling,focusing on a number of newly identified proteins involved in mitochondrial Ca2+ uptake and release. We further discuss our recent findings,revealing how mitochondrial Ca2+ influences the matrix redox state. As a result,mitochondrial Ca2+ is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease.     

甘草水溶物诱导MGC-803细胞凋亡中胞内Ca~(2+)、pH与线粒体△ψ_m的变化

Changes of intracellular Ca 2+ ,pH value and mitochondria membrane potential(△Ψ m) in the apoptosis of MGC 803 cells induced by water soluble constituents of Glycyrrhiza uralensis Fisch(WSCG) were investigated.MGC 803 cells were incubated with 0.5,0.75,1.0 and 1.5 g/L WSCG for 1,4,7,11,15,19 and 23 hours respectively.The percentage of apoptosis and intracellular Ca 2+ content increased in a dose and time dependent manner,except that the intracellular Ca 2+ content of 1.5 g/L WSCG treated cells started to decrease after 11 hours.The cells were alkalized after various treatments,except that 1.5 g/L WSCG treated cells turned to acidify after 11 hours.It was found that the mitochondria △Ψ m of all treated cells decreased drastically at the first hour,then kept decreasing slowly until the 23rd hour.The results indicated that intracellular Ca 2+ ,pH and mitochondria △Ψ m all played pivotal roles in the apoptosis of MGC 803 cells induced by WSCG.     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

Mitochondrial Ca(2+)homeostasis in the regulation of apoptotic and necrotic cell deaths

Using distinct models of apoptosis and necrosis, we have investigated the effect of mitochondrial Ca(2+)(Ca(m)) homeostasis in the regulation of cell death in neuroblastoma cells as well as cardiac myocytes. The steady state level of Ca(m)was determined as the FCCP-releasable Ca(2+). Culturing cells with low concentration of extracellular Ca(2+)(Ca(o)) or with EGTA triggered an early reduction in both the Ca(m)store and the membrane potential (DeltaPsi(m)). This was followed by the detection of cytochrome c release, caspase activation, and apoptosis. Inhibitors of the mitochondrial permeability transition pore such as cyclosporin A and Bcl-2 blocked the release of Ca(m)and inhibited apoptosis. In contrast, mitochondrial Ca(2+)overload resulted in necrotic cell death. Culturing cells in the presence of excess Ca(o)led to increased Ca(m)load together with a decrease of DeltaPsi(m)that reached maximum at 1 h, with necrosis occurring at 2 h. While the decline of Ca(m)and DeltaPsi(m)was a coupled reaction for apoptosis, this relationship was uncoupled during necrosis. Clonazepam, a relatively specific inhibitor of the mitochondrial Na/Ca exchanger, was able to protect the cells from necrosis by reducing Ca(m)overload. Importantly, combination of clonazepam and cyclosporin showed a cooperative effect in further reducing the Ca(m)overload and abolished cell death. The data imply the participation of Ca(m)homeostasis in the regulation of apoptosis and necrosis.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

机械刺激诱导的烟草悬浮细胞一氧化氮产生途径及其与C矿和钙调素的关系

通过提高摇床转速对烟草细胞施加机械刺激（Ms）可诱导其胞内一氧化氮（No）的快速产生和一氧化氮合酶（Nos）活性的提高,这种MS诱导的NO产生可被N0清除剂cPTIO和NOS抑制剂L-NMMA显著抑制。此外,Ca2＋螯合剂EGTA、质膜Ca＋通道阻断剂La3＋、胞内Ca2＋通道拮抗剂钌红,以及钙调素抑制剂CPZ和TFP预处理均不同程度地抑制了机械刺激诱导的烟草细胞NO的产生,而机械刺激过程中钙调素活性显著上升并与NOS活性和NO含量的变化相一致。这些结果暗示着（类）Nos酶催化的精氨酸依赖途径可能是机械刺激诱发烟草细胞NO产生的主要途径,Ca2＋／CAM可能通过调节（类）NOS活性来调控No的产生。