TREK-1通道活性改变对大鼠局灶性脑缺血后细胞凋亡和凋亡相关蛋白的影响

目的:观察双孔钾通道TREK-1活性改变对大鼠局灶性脑缺血后细胞凋亡和凋亡相关蛋白的影响。方法:45只大鼠随机分为假手术组(10只)、对照组(10只)和干预组(25只)。建立大鼠光化学脑缺血模型,假手术组不注射玫瑰红,干预组侧脑室注射不同浓度(100μmol/L、250μmol/L、500μmol/L、1 mmol/L)亚麻酸(LIN),对照组注射等量生理盐水。应用免疫荧光双标法观察正常生理情况下TREK-1在大脑神经细胞中的表达,TUNEL及DAPI双标法检测缺血边缘区细胞凋亡,Western blot法检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸化细胞外信号调节激酶(p-erk)表达。结果:正常生理情况下,TREK-1在神经元和星形胶质细胞中均有表达。与假手术组相比,对照组大量细胞凋亡,Bcl-2/Bax值下降,p-erk蛋白增加(P<0.05);与对照组比较,干预组细胞凋亡显著减少,Bcl-2/Bax值上升,p-erk蛋白降低(P<0.05)。结论:TREK-1在神经元和星形胶质细胞中均有表达。TREK-1激动剂LIN可显著抑制脑缺血后细胞凋亡,上调Bcl-2与Bax比值,抑制erk磷酸化。     

线粒体分裂蛋白抑制剂对β淀粉样蛋白诱导小胶质细胞凋亡的作用及机制

目的:研究线粒体分裂蛋白抑制剂对β淀粉样蛋白(Aβ)诱导小胶质细胞凋亡的作用及其机制。方法:随机将BV-2小胶质细胞分为con组、Aβ组、mdi组和Aβ+mdi组,con组不做特殊处理,mdi组培养基中加入10μmol/L mdivi-1,Aβ组培养基中加入20μmol/L Aβ,Aβ+mdi组培养基分别加入2、5、10、20μmol/L mdivi-1和20μmol/L Aβ。采用MTT法检测细胞存活率,TUNEL染色检测细胞凋亡,Western blot法检测Drp1、CytC和Caspase-3蛋白水平变化,RT-PCR法检测CA11b mRNA表达变化。结果:与con组相比,Aβ组的细胞存活率明显下降,凋亡显著增加,CA11b mRNA上升,线粒体Drp1、细胞浆CytC和激活的Caspase-3增加,差异有统计学意义(P<0.05);与Aβ组相比,Aβ+mdi组的细胞存活率明显上升,凋亡显著减少,CA11b mRNA下降,线粒体Drp1、细胞浆CytC和激活的Caspase-3减少,差异有统计学意义(P<0.05)。结论:线粒体分裂蛋白抑制剂对Aβ诱导小胶质细胞凋亡有保护作用,其机制可能为抑制线粒体/CytC/Caspase-3凋亡途径。     

Drp1介导的线粒体动力学平衡在脓毒症心肌细胞损伤的机制研究北大核心CSCD

目的分析抑制动态相关蛋白1(dynamin related protein 1,Drp1)介导的线粒体过度分裂对脓毒症心肌细胞和线粒体功能的影响,探讨维持线粒体动力学平衡在脓毒症心肌病(sepsis induced cardiomyopathy,SIC)发病过程中的保护作用。方法培养大鼠H9C2心肌细胞,脂多糖(lipopolysaccharide,LPS)刺激细胞建立SIC细胞模型,LPS刺激30 min前予线粒体分裂抑制剂(mitochondrial division inhibitor 1,Mdivi-1)干预,分为对照组(Control)、LPS刺激组(LPS)Mdivi-1对照组(Mdivi-1)和LPS+Mdivi-1干预组(LPS+Mdivi-1)。CCK-8检测细胞存活率,乳酸脱氢酶(lactate dehydrogenase,LDH)检测细胞损伤情况,MitoTracker探针染色激光共聚焦观察线粒体形态,JC-1探针染色检测线粒体膜电位水平,DCFH-DA探针检测细胞总活性氧(ROS)水平,Annexin V-FITC/PI探针流式细胞仪检测细胞凋亡情况,实时荧光定量PCR、Western blot检测Drp1、视神经萎缩蛋白1(optic Atrophy 1,Opa1)、线粒体融合蛋白2(mitofusin 2,Mfn2)表达水平。组间差异比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与Control组相比,LPS刺激组细胞存活率明显降低,LDH活性升高,线粒体平均长度减小,线粒体膜电位下降、细胞ROS生成增多,细胞凋亡增加(均P<0.05);予以Mdivi-1干预后,与LPS刺激组相比,细胞存活率升高,心肌细胞损伤减轻线粒体平均长度延长,线粒体功能障碍减轻,细胞凋亡受到抑制(均P<0.05)。结论Mdivi-1可能通过抑制Drp1介导的线粒体分裂维持线粒体动力学平衡,减轻线粒体功能障碍,从而保护LPS诱导的心肌细胞损伤。     

Drp1介导的线粒体动力学平衡在脓毒症心肌细胞损伤的机制研究

目的分析抑制动态相关蛋白1(dynamin related protein 1, Drp1)介导的线粒体过度分裂对脓毒症心肌细胞和线粒体功能的影响, 探讨维持线粒体动力学平衡在脓毒症心肌病(sepsis induced cardiomyopathy, SIC)发病过程中的保护作用。方法培养大鼠H9C2心肌细胞, 脂多糖(lipopolysaccharide, LPS)刺激细胞建立SIC细胞模型, LPS刺激30 min前予线粒体分裂抑制剂(mitochondrial division inhibitor 1, Mdivi-1)干预, 分为对照组(Control)、LPS刺激组(LPS)、Mdivi-1对照组(Mdivi-1)和LPS+Mdivi-1干预组(LPS+Mdivi-1)。CCK-8检测细胞存活率, 乳酸脱氢酶(lactate dehydrogenase, LDH)检测细胞损伤情况, MitoTracker探针染色激光共聚焦观察线粒体形态, JC-1探针染色检测线粒体膜电位水平, DCFH-DA探针检测细胞总活性氧(ROS)水平, Annexin V-FITC/PI探针流式细胞...     

C57BL/6小鼠心肺复苏后心肌细胞线粒体自噬及细胞凋亡的相互作用与调节机制的研究

目的:探讨在心搏骤停心肺复苏系统性缺血再灌注损伤过程中,C57BL/6小鼠心室肌细胞线粒体自噬与细胞凋亡之间的相互作用和调控机制。方法:80只健康雄性C57BL/6小鼠应用高钾合并窒息法制备心搏骤停心肺复苏模型。其中6只复苏前组,74只小鼠造模。造模小鼠中有50只自主循环恢复(ROSC),将其随机分为复苏后2、12、24、48h组。应用透射电镜检测心肌细胞线粒体形态;应用Western blot检测线粒体自噬蛋白表达和蛋白磷酸化表达,检测与线粒体相关的细胞凋亡信号通路的蛋白表达和蛋白磷酸化表达。结果:C57BL/6高钾心搏骤停心肺复苏小鼠模型在复苏后48h内线粒体自噬特异性信号蛋白LC3-II表达持续显著增加,启动线粒体自噬的蛋白ULK1表达也持续增加。在复苏后12、24、48hC57BL/6小鼠通过mTOR磷酸化ULK1抑制线粒体自噬过度增加。C57BL/6小鼠在复苏后2、12h凋亡信号caspase-9蛋白活化增加,复苏后24、48h caspase-9蛋白激活逐步减少。心肺复苏后抑制凋亡因子Bcl-2未被明显激活。蛋白细胞色素C(Cyt c)和Smac/Diablo是线粒体释放的促进细胞凋亡的信号。C57BL/6小鼠在复苏后48h内线粒体释放的Smac/Diablo未见显著增加,线粒体释放的Cyt c在复苏后12h达到峰值。结论:免疫功能正常的C57BL/6小鼠心肌细胞在心搏骤停心肺复苏系统性缺血再灌注损伤过程中,激活线粒体自噬,清除受损的线粒体。在复苏后24h抑制线粒体自噬的信号通路也被激活,提示体内的调控机制发挥作用,防止线粒体过度自噬。线粒体释放促进细胞凋亡的信号蛋白Cyt c在复苏后12h达到峰值。Cyt c与procaspase-9/Apaf 1交联,促使caspase-9活化。     

脂蟾毒配基对人胃癌BGC-823细胞系细胞生长的干预效应

目的:观察中药蟾酥主要成分之一——脂蟾毒配基对体外培养人胃癌BGC-823细胞系细胞生长的干预及其与细胞色素C和半胱氨酸天冬氨酸蛋白酶3活化的关系。方法:实验于2004-09/2005-12在北京大学医学部细胞生物学系实验室完成。体外培养人胃癌BGC-823细胞系,脂蟾毒配基各组加入不同浓度的脂蟾毒配基(先用二甲基亚砜溶解,再经RPMI-1640培养液稀释至终浓度。细胞增殖及增殖抑制实验、细胞核形态观察及核DNA含量测定实验中脂蟾毒配基浓度分别为0.1,1,10μmol/L;细胞周期分布实验、细胞凋亡率实验、线粒体膜电位实验中脂蟾毒配基浓度分别为0,1,2.5,5μmol/L)。空白对照组加入RPMI-1640培养液。盐酸阿克拉霉素组加入5μmol/L盐酸阿克拉霉素。采用酸性磷酸酶法检测细胞增殖抑制作用;细胞荧光分光光度仪观察细胞核形态及核DNA含量测定;流式细胞仪检测细胞周期、细胞凋亡率及其线粒体膜电位变化;Westernblot检测与细胞凋亡相关基因蛋白表达的变化。结果:①经0.1,1,10μmol/L脂蟾毒配基分别作用24,48,72h后,细胞生长显著抑制,脂蟾毒配基对癌细胞的生长抑制百分率与剂量、时间呈正相关,其IC50分别为3.9,2.0,1.5μmol/L。②随脂蟾毒配基作用浓度和时间的延长,癌细胞核荧光强度减弱,细胞核DNA含量明显降低。③0.1,1μmol/L脂蟾毒配基作用24～48h后癌细胞阻滞在S期,5μmol/L脂蟾毒配基作用72h时,细胞阻滞在G2 M期;盐酸阿克拉霉素与癌细胞作用24～48h,细胞阻滞在S期,作用72h时细胞阻滞在G2 M期。④脂蟾毒配基作用后,细胞凋亡率明显高于对照组。⑤脂蟾毒配基引起细胞线粒体膜电位下降,释放入胞质中的细胞色素C增多,促进半胱氨酸天冬氨酸蛋白酶3蛋白活化,抑制Bcl-2蛋白表达,诱导细胞凋亡。结论:体外实验条件下,脂蟾毒配基抑制人胃癌BGC-823细胞生长并诱导细胞发生凋亡,其诱导凋亡作用机制与线粒体通路有关。     

菊花活性成分Parthenolide对人鼻咽癌CNE2细胞线粒体功能和半胱天冬酶活性的影响

背景鼻咽癌是我国南方重点防治的恶性肿瘤之一,探讨天然草本菊花活性成分在NPC化学预防的作用,对建立现代康复体系具有重要意义.目的研究菊花活性成分倍半萜烯内酯(SLs)对人鼻咽癌细胞中线粒体功能和半胱天冬酶活化途径的影响.设计完全随机对照分组设计.地点和对象由中山大学公共卫生学院预防医学系完成,对象为低分化CNE2细胞株.干预对parthenolide(PN)进行剂量-反应和时间效应作用观察,采用噻唑蓝(MTT)颜色反应检测细胞线粒体功能,底物荧光光谱法测定细胞内caspase-9和-3活性,蛋白免疫印迹法检测线粒体内细胞色素C释放和caspase-3酶原降解片段;并应用特异性抑制剂进行细胞内cas-pase途径阻断实验.主要观察指标细胞线粒体功能、细胞内caspase-9和caspase-3活性、线粒体细胞色素C的释放和caspase-3酶原的降解.结果PN(1～100μmol/L)作用12 h和2 4h后,MTT颜色反应抑制率随剂量显著增高,呈明显剂量依赖性(Pearson's r=0.7322,0.7703,P＜0.05),IC50分别为252.94μmol/L和49.63 μmol/L;但caspase-9和-3活性未见升高,未见细胞色素C释放和caspase-3酶原降解片段形成.PN与caspase抑制剂联合作用后,caspase-9和-3活性明显低于对照和单独PN作用(t=9.146,8.280,27.325,27.450,P＜0.05).结论PN可明显诱导线粒体功能降低和丢失,与介导肿瘤细胞增殖抑制和毒性效应有关,但不影响CNE2细胞caspase途径的活化,此项研究为NPC的化学预防和康复提供依据.     

淀粉样β蛋白25～35诱导的PC12细胞凋亡与奥氮平的保护作用

背景:在阿尔茨海默病中起着重要作用的淀粉样β蛋白能够诱导细胞凋亡。目的:探讨奥氮平对淀粉样β蛋白25～35诱导的PC12细胞凋亡的机制及其保护作用。单位:解放军总医院南楼神经科。设计:随机设计。材料:实验于2002-05/2003-03在加拿大萨斯卡彻温大学医学院神经精神研究所完成。方法:P12细胞在RPMI1640培养液中培养。在96孔板每孔中接种100μL细胞悬液,在胶原被覆的25cm2培养瓶中接种5mL细胞悬液,培养24h后,分别加50μmol/L、100μmol/L奥氮平培养24h,再加不同浓度的淀粉样β蛋白25～35(0.01μmol/L、2μmol/L、20μmol/L)培养24h。将收获好的96孔板以淀粉样β蛋白25～35诱导PC12细胞凋亡,采用MTT比色分析测定细胞存活率。收获25cm2培养瓶中的PC12细胞,应用Westernblot检测奥氮平对PC12细胞Bax、半胱氨酸天冬氨酸蛋白酶3表达的影响。主要观察指标:细胞存活率的测定,PC12细胞中Bax、半胱氨酸天冬氨酸蛋白酶3的表达水平。结果:①细胞存活率比较:淀粉样β蛋白25～35诱导的PC12细胞的细胞活性从75%降低到35%;50μmol/L、100μmol/L奥氮平预处理组PC12细胞的活性明显提高。②奥氮平对淀粉样β蛋白25～35诱导的PC12细胞凋亡中Bax表达的影响:0.01μmol/L,2μmol/L,20μmol/L淀粉样β蛋白25～35处理的PC12细胞Bax的表达增加,50μmol/L奥氮平预处理使淀粉样β蛋白25～35诱导的PC12细胞Bax的表达减低。③奥氮平对Aβ25～35诱导的PC12细胞凋亡中半胱氨酸天冬氨酸蛋白酶3表达的影响:0.001μmol/L、0.01μmol/L淀粉样β蛋白处理的PC12无变化,50μmol/L奥氮平预处理对PC12细胞表达亦无影响;2μmol/L、20μmol/L淀粉样β蛋白25～35处理的PC12细胞表达增高,50μmol/L奥氮平预处理能抑制其表达升高。结论:①淀粉样β蛋白25～35能够诱发与细胞凋亡密切相关的Bax、半胱氨酸天冬氨酸蛋白酶3在培养的PC12细胞中高表达。②奥氮平具有降低其表达,提高PC12细胞存活的保护作用。     

细胞周期抑制剂对糖氧剥夺诱导神经元凋亡的保护机制研究

目的:探讨细胞周期抑制剂Roscovitine(Ros)对糖氧剥夺(OGD)诱导的鼠大脑皮质神经元凋亡的保护作用及可能机制。方法:体外培养大鼠皮质神经元,随机分为对照组、OGD1h后恢复糖氧供给(OGD/R)3h、6h、12h、24h组及Ros(100μM)组。Western Blot检测各组神经元磷酸化视网膜母细胞瘤蛋白(p-Rb)和E2F1的表达情况;免疫荧光细胞化学染色观察OGD/R12h组及Ros组神经元p-Rb表达;TUNEL法检测OGD/R12h组及Ros组神经元凋亡情况。结果:OGD/R各组神经元p-Rb及E2F1的表达均较对照组增高(P<0.05),12h达最高;Ros组p-Rb及E2F1的表达减少,少于OGD/R12h组(P<0.05);Ros组p-Rb和TUNEL阳性细胞率均低于OGD/R 12h组(P<0.01),两组中大部分TUNEL阳性细胞与p-Rb表达共定位。结论:Ros可能通过抑制Rb磷酸化及E2F1介导的凋亡机制来减少缺血缺氧后的神经元凋亡。     

姜黄素对UMI-77诱导急性T淋巴细胞白血病细胞凋亡的影响及其相关机制研究

目的:探究姜黄素对Mcl-1小分子抑制剂UMI-77诱导的人急性T淋巴细胞白血病(T-ALL)细胞凋亡的影响及其相关机制。方法:培养T-ALL细胞系Molt-4,用不同浓度的姜黄素和Mcl-1小分子抑制剂UMI-77分别处理细胞24 h, MTT法检测经不同浓度姜黄素和UMI-77分别处理后的细胞存活率;根据姜黄素和UMI-77的作用浓度,实验设置对照、姜黄素(20μmol/L姜黄素处理细胞)、UMI-77组(15μmol/L Mcl-1小分子抑制剂UMI-77处理细胞)和姜黄素+UMI-77(20μmol/L姜黄素加15μmol/L Mcl-1小分子抑制剂UMI-77处理细胞)共4组,MTT法检测细胞增殖抑制率,Annexin V-FITC/PI双染法和TUNEL染色检测细胞的凋亡情况,DCFH-DA探针检测细胞活性氧,JC-1荧光探针检测线粒体膜电位,Western blot检测细胞凋亡相关蛋白和Notch1信号通路相关蛋白的表达水平。结果:经过不同浓度的姜黄素和Mcl-1小分子抑制剂UMI-77处理Molt-4细胞后,细胞存活率降低(P<0.05);与对照组比较,姜黄素组和...     

Oligomycin and antimycin A prevent nitric oxide-induced apoptosis by blocking cytochrome C leakage

Nitric oxide (NO) is a potent inducer of apoptosis, and its cytotoxicity is closely related to mitochondrial dysfunction. In this study we investigated the effects of a F0F1-ATPase inhibitor, oligomycin, and a mitochondrial respiratory chain complex III inhibitor, antimycin A, on NO-induced apoptosis. We used a normal rat gastric-epithelium cell line, RGM-1, treated with a pure NO donor, NOC-1 —1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene — in the presence or absence of oligomycin or antimycin A. Changes in the expressions of Bax or Bcl-2 proteins, release of cytochrome C from mitochondria into the cytosol, activation of caspase-3, and changes in the mitochondrial membrane potential (ΔΨ) were measured with the use of Western blotting, c43 lorimetric assays, and a mitochondrial potential sensor, JC-1 dye. Treatment with NOC-18 induced dose-dependent apoptotic cell death in RGM-1 cells. Cell death was accompanied by mitochondrial depolarization, increases in Bax protein expression and cytochrome C leakage, and, subsequently, caspase-3 activation. Oligomycin and antimycin A prevented NO-induced apoptosis in a dose-dependent fashion by preventing cytochrome C release independent of Bcl-2 expression. However, neither compound affected the up-regulation of Bax protein. On the one hand, oligomycin treatment was not accompanied by a decline in ΔΨ. On the other hand, antimycin A treatment decreased ΔΨ regardless of NOC-18 treatment. The findings of this study suggest that various functional molecules that constitute the mitochondrial respiratory chain may contribute to cytochrome C release that occurs during NO-induced apoptosis.     

银杏叶提取物对神经元缺糖缺氧/复糖复氧性损伤的作用

目的研究银杏叶提取物(EGB761)对体外培养神经元缺糖缺氧/复糖复氧性损伤的作用,并探讨其作用机制。方法利用体外培养的皮层神经元,通过去除培养液中的葡萄糖和氧气(oxygen andglucose deprivation,OGD)模拟缺血缺氧,恢复糖氧供给模拟再灌流。再灌流时分三组加入不同浓度的EGB761观察其作用。噻唑盐比色法(MTT)测定细胞活性,碘化丙锭(PI)与Hoechst33258双染色观察神经元凋亡,免疫蛋白印迹法测定Bcl-2、Bax蛋白的表达。结果EGB761可减少缺糖缺氧/复糖复氧导致的神经元凋亡,提高因该损伤而下降的MTT值以及Bcl-2蛋白的表达。Bax蛋白在EGB761处理前后均无明显变化。结论EGB761可拮抗缺糖缺氧/复糖复氧导致的神经元凋亡,并且该作用可能与诱导Bcl-2蛋白表达有关。     

Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation

Silymarin, a plant flavonoid, has been shown to inhibit skin carcinogenesis in mice. However, the mechanism responsible for the anti-skin carcinogenic effects of silymarin is not clearly understood. Here, we report that treatment of JB6 C141 cells (preneoplastic epidermal keratinocytes) and p53+/+ fibroblasts with silymarin and silibinin (a major constituent of silymarin) resulted in a dose-dependent inhibition of cell viability and induction of apoptosis in an identical manner. Silymarin-induced apoptosis was determined by fluorescence staining (8-64% apoptosis) and flow cytometry (12-76% apoptosis). The silymarin-induced apoptosis was primarily p53 dependent because apoptosis occurred to a much greater extent in the cells expressing wild-type p53 (p53+/+, 9-61%) than in p53-deficient cells (p53-/-, 6-20%). The induction of apoptosis in JB6 C141 cells was associated with increased expression of the tumor suppressor protein, p53, and its phosphorylation at Ser15. The constitutive expression of antiapoptotic proteins Bcl-2 and Bcl-xl were decreased after silymarin treatment, whereas the expression of the proapoptotic protein Bax was increased. There was a shift in Bax/Bcl-2 ratio in favor of apoptotic signal in silymarin-treated cells, which resulted in increased levels of cytochrome c release, apoptotic protease-activating factor-1, and cleaved caspase-3 and poly(ADP-ribose) polymerase in JB6 C141 cells. The shift in Bax/Bcl-2 ratio was more prominent in p53+/+ fibroblasts than in p53-/- cells. Silymarin-induced apoptosis was blocked by the caspase inhibitor (Z-VAD-FMK) in JB6 C141 cells which suggested the role of caspase activation in the induction of apoptosis. These observations show that silymarin-induced apoptosis is primarily p53 dependent and mediated through the activation of caspase-3.     

Inhibition of Apoptosis in Chlamydia-infected Cells: Blockade of Mitochondrial Cytochrome c Release and Caspase Activation

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-α, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.     

The cyclooxygenase-2 inhibitor celecoxib blocks phosphorylation of Akt and induces apoptosis in human cholangiocarcinoma cells

The expression of cyclooxygenase (COX)-2 is increased in human cancers including cholangiocarcinoma. This study was designed to evaluate the effect and mechanisms of the selective COX-2 inhibitor celecoxib in the growth control of human cholangiocarcinoma cells. Immunohistochemical analysis using human cholangiocarcinoma tissues showed increased levels of COX-2 as well as phospho-Akt (Thr (308)), a protein kinase activated by COX-2-mediated prostaglandins, in human cholangiocarcinoma cells. Treatment of cultured human cholangiocarcinoma cells (HuCCT1, SG231, and CCLP1) with celecoxib resulted in a dose- and time-dependent reduction of cell viability. Fluorescence microscopy, Western blot, and caspase activity assays demonstrated that celecoxib induced morphological features of apoptosis, activation of caspase-9 and caspase-3, and release of cytochrome c. The celecoxib-induced cell death was significantly blocked by N-benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone, a wide-spectrum caspase inhibitor. Furthermore, cholangiocarcinoma cells treated with celecoxib showed significant reduction of Akt phosphorylation, whereas the levels of Bcl-2 and Bax were not altered. Inhibition of Akt activation by LY294002 significantly decreased the viability of human cholangiocarcinoma cells. These findings suggest that celecoxib inhibits cholangiocarcinoma growth partly through induction of apoptosis and inhibition of Akt phosphorylation.     

Combination treatment with arsenic trioxide and phytosphingosine enhances apoptotic cell death in arsenic trioxide-resistant cancer cells

Resistance to anticancer drugs can sometimes be overcome by combination treatment with other therapeutic drugs. Here, we showed that phytosphingosine treatment in combination with arsenic trioxide (As(2)O(3)) enhanced cell death of naturally As(2)O(3)-resistant human myeloid leukemia cells. The combination treatment induced an increase in intracellular reactive oxygen species level, mitochondrial relocalization of Bax, poly(ADP-ribose) polymerase-1 (PARP-1) activation, and cytochrome c release from the mitochondria. N-acetyl-l-cysteine, a thiol-containing antioxidant, completely blocked Bax relocalization, PARP-1 activation, and cytochrome c release. Pretreatment of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, a PARP-1 inhibitor, or PARP-1/small interfering RNA partially attenuated cytochrome c release, whereas the same treatment did not affect Bax relocalization. The combination treatment induced selective activation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK by treatment of SB203580 or expression of dominant-negative forms of p38 MAPK suppressed the combination treatment-induced Bax relocalization but did not affect PARP-1 activation. In addition, antioxidant N-acetyl-l-cysteine completely blocked p38 MAPK activation. These results indicate that phytosphingosine in combination with As(2)O(3) induces synergistic apoptosis in As(2)O(3)-resistant leukemia cells through the p38 MAPK-mediated mitochondrial translocation of Bax and the PARP-1 activation, and that p38 MAPK and PARP-1 activations are reactive oxygen species dependent. The molecular mechanism that we elucidated in this study may provide insight into the design of future combination cancer therapies to cells intrinsically less sensitive to As(2)O(3) treatment.     

制备神经细胞实验性缺氧缺糖模型的简易方法

背景:在神经细胞培养中实验性缺氧缺糖在一定程度上模拟缺血性卒中,对于研究缺血性神经元损伤的进程和病理生理学机制有非常重要的用处.目的:在神经元培养时制作实验性缺氧缺糖模型.设计、时间及地点:分组对照观察,实验于2007-01/2008-03在北京大学第三医院中心实验室完成. 材料:17-19 d胎龄的Wistar大鼠.方法:细胞培养取17～19 d胎龄的Wistar大鼠的皮质神经元做原代细胞培养,并且去掉污染的非神经原细胞.缺氧缺糖的诱导分为3组:实验组将第7天的皮质神经元置于无糖甲衡盐溶液和2%去氧酶中,在37℃的潮湿保温箱中培育.空白对照组培养基为含20 mmol/L葡萄糖的无去氧酶平衡盐溶液.假性实验组培养基为含20 mmol/L葡萄糖和失活的去氧酶平衡盐溶液.主要观察指标:以血气分析进行氧浓度的测定;以相差显微镜观察实验组培养细胞神经元死亡状况;以用乳酸脱氢酶检测盒检测乳酸脱氢酶活性;以锥虫蓝染色观察缺氧缺糖对神经元存活力的影响.结果:氧浓度测定显示在加入去氧酶后培养基迅速产生缺氧状态;乳酸脱氢酶检测显示在用去氧酶和无糖平衡盐处理后,培养基中乳酸脱氢酶释放显著增加;锥虫蓝染色和相差显微镜检查显示经去氧酶和无糖平衡盐处理后实验组的细胞活力明显下降,大部分神经元在6 h死亡.结论:实验结果显示去氧酶与无精平衡盐液可联合用于神经元培养时产生缺氧缺糖状态,其在体外模拟脑缺血的相关研究中有重要作用.     

Apoptosis Induced by Microbubble-Assisted Acoustic Cavitation in K562 Cells: The Predominant Role of the Cyclosporin A-Dependent Mitochondrial Permeability Transition Pore

Acoustic cavitation of microbubbles has been described as inducing tumor cell apoptosis that is partly associated with mitochondrial dysfunction; however, the exact mechanisms have not been fully characterized. Here, low-intensity pulsed ultrasound (1 MHz, 0.3-MPa peak negative pressure, 10% duty cycle and 1-kHz pulse repetition frequency) was applied to K562 chronic myelogenous leukemia cells for 1 min with 10% (v/v) SonoVue microbubbles. After ultrasound exposure, the apoptotic index was determined by flow cytometry with annexin V–fluorescein isothiocyanate/propidium iodide. In addition, mitochondrial membrane potential (ΔΨ_m) was determined with the JC-1 assay. Translocation of apoptosis-associated protein cytochrome c was evaluated by Western blotting. We found that microbubble-assisted acoustic cavitation can increase the cellular apoptotic index, mitochondrial depolarization and cytochrome c release in K562 cells, compared with ultrasound treatment alone. Furthermore, mitochondrial dysfunction and apoptosis were significantly inhibited by cyclosporin A, a classic inhibitor of the mitochondrial permeability transition pore; however, the inhibitor of Bax protein, Bax-inhibiting peptide, could not suppress these effects. Our results suggest that mitochondrial permeability transition pore opening is involved in mitochondrial dysfunction after exposure to microbubble-assisted acoustic cavitation. Moreover, the release of cytochrome c from the mitochondria is dependent on cyclosporin A–sensitive mitochondrial permeability transition pore opening, but not formation of the Bax-voltage dependent anion channel complex or Bax oligomeric pores. These data provide more insight into the mechanisms underlying mitochondrial dysfunction induced by acoustic cavitation and can be used as a basis for therapy.     

氯离子通道阻断剂NPPB对顺铂诱导C6细胞损伤的作用

目的探讨氯离子通道阻断剂NPPB对顺铂(DDP)诱导的神经胶质瘤C6细胞损伤中的作用。方法应用MTT法检测C6细胞的生存率;RT-PCR检测Bcl-2、Bax及ClC-3的mRNA表达;Western blotting方法检测Bcl-2、Bax、Cyt-C的蛋白水平。结果与对照组相比,NPPB组C6细胞生存率、Bax/Bcl-2比值、ClC-3 mRNA及Cyt-C蛋白表达无明显变化;DDP组C6细胞生存率明显下降(P<0.05),ClC-3 mRNA表达降低,Bax/Bcl-2比值及Cyt-C蛋白表达明显增高。而DDP与NPPB联合组C6细胞生存率与DDP组相比明显增高,ClC-3 mRNA表达增高,Bax/Bcl-2比值呈下降趋势,Cyt-C蛋白表达明显降低。结论氯离子通道阻断剂NPPB可能通过降低Bax/Bcl-2比值,抑制Cyt-C的释放而拮抗DDP对肿瘤细胞增殖的抑制作用。