细胞凋亡的检测方法

细胞凋亡（apoptosis）即程序性细胞死亡（programmed cell death,PCD）是细胞在一定的生理或病理状态下,遵循自身的程序,自己结束自己生命的过程,最后细胞脱落离体或裂解为若干凋亡小体,而被其他细胞吞噬。它是由基因控制的、高度有序的细胞主动死亡过程,不导致炎症反应。如果细胞凋亡发生紊乱,会引起机体疾病的发生,当然它也参与机体许多病理生理过程。近年来,由于实验模型及检测方法的完善,细胞凋亡的研究获得了重大突破,受到医学各个领域的关注,现就关于细胞凋亡的研究方法作一下综述。     

氯化钴对PC12细胞的凋亡作用

目的　确定氯化钴(CoCl2 )对PC12细胞的影响。方法　构建CoCl2 诱导的PC12细胞模型,检测CoCl2 对PC12细胞的毒性作用;将Caspases的抑制基因p3 5转染PC12细胞得到可稳定表达p3 5基因的细胞株PC12 p3 5 ,检测p3 5对CoCl2 诱导的PC12细胞的作用;检测Caspases特异性多肽抑制剂Z VAD FMK对CoCl2 诱导的PC12细胞的作用。结果　分别以10 0、3 0 0、5 0 0、70 0和10 0 0 μmol LCoCl2 诱导PC12细胞2 4h或以5 0 0 μmol LCoCl2 分别诱导PC12细胞12、2 4、3 6、48和60h后,PC12细胞存活率均明显下降(P <0 0 1) ,并与CoCl2 诱导的时间和浓度呈正相关;细胞亚显微结构检测,流式细胞分析和DNA片段化结果也表明5 0 0 μmol LCoCl2 诱导PC12细胞2 4h可使PC12细胞出现明显凋亡特征。构建可稳定表达Caspase蛋白酶抑制基因p3 5的PC12细胞株,或分别加入5 0和10 0 μmol LCaspases多肽抑制剂Z VAD FMK预处理PC12细胞1h后,以5 0 0 μmol LCoCl2 诱导PC12细胞2 4h ,形态学观察结果,细胞存活率检测和流式细胞分析均表明p3 5基因和Z VAD FMK均可有效地抑制CoCl2 诱导的PC12细胞凋亡(P <0 0 1)。结论　CoCl2 可诱导PC12细胞凋亡。     

人肾透明细胞癌中细胞凋亡相关因子Caspase-3和Caspase-9的表达

目的研究人肾透明细胞癌组织中细胞凋亡相关重要因子Caspase-3、Caspase-9表达的变化与肾癌的关系。方法标本离体后分成肾透明细胞癌组、癌旁组织组、正常肾组织组,均用10%的甲醛溶液固定48h,常规石蜡包埋、切片5μm,应用免疫组织化学方法进行观察研究。结果 Caspase-3与Caspase-9均在正常肾组织中可见少量弱阳性反应,癌旁组织阳性表达增加,肾癌组织表达明显减少,两两比较结果显示,3组之间差异均有统计学意义（P〈0.01）。肾癌4个期之间比较差异无统计学意义（P〉0.05）。Caspase-3与Caspase-9蛋白表达呈正相关（r=0.988,P〈0.05）。结论癌组织中Caspase-3和Caspase-9表达下降,说明细胞凋亡减少,癌组织细胞增殖旺盛,是肿瘤形成的重要机制。     

细胞凋亡与心力衰竭

细胞凋亡是机体维持稳定的一个重要生理过程,受到基因的调控并由细胞自主进行.蛋白水解酶是凋亡过程的主要参与者,其中最重要的是半胱氨酸天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase)家族.根据凋亡过程是否依赖半胱氨酸天冬氨酸蛋白水解酶,细胞凋亡可分为3种途径,包括死亡受体途径、线粒体途径和内质网途径.细胞对凋亡存在正向和反向两种调节机制,从而精确控制着凋亡的反应程度.细胞凋亡对衰竭的心脏有显著的结构和功能性影响,抗凋亡干预疗法对于改善心力衰竭具有重要的临床意义.     

细胞凋亡与心力衰竭

细胞凋亡是机体维持稳定的一个重要生理过程,受到基因的调控并由细胞自主进行。蛋白水解酶是凋亡过程的主要参与者,其中最重要的是半胱氨酸天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase)家族。根据凋亡过程是否依赖半胱氨酸天冬氨酸蛋白水解酶,细胞凋亡可分为3种途径,包括死亡受体途径、线粒体途径和内质网途径。细胞对凋亡存在正向和反向两种调节机制,从而精确控制着凋亡的反应程度。细胞凋亡对衰竭的心脏有显著的结构和功能性影响,抗凋亡干预疗法对于改善心力衰竭具有重要的临床意义。     

丙泊酚通过抑制线粒体途径的细胞凋亡保护心肌缺血再灌注损伤(英文)

目的观察丙泊酚对离体大鼠心肌缺血再灌注(I-R)损伤的影响,并从线粒体过氧化损伤方面探讨其可能的作用机制。方法应用Langendorff离体心脏灌流系统,全心停灌25 min,再灌注30 min建立心肌I-R损伤模型。记录各项心功能指标;测定心肌线粒体呼吸链的完整性、膜肿胀度和丙二醛(MDA)含量;流式细胞术检测心肌细胞凋亡率及Bcl-2和Bax蛋白的表达,免疫组化法测定半胱氨酸天冬氨酸蛋白酶(caspase)-3,-9和-8的表达。结果与I-R组相比,缺血前10 min开始,并于再灌注期间持续灌流丙泊酚30和60μmol·L~(-1)能明显改善I- R后的心功能;心肌线粒体呼吸链损伤有所恢复,膜肿胀度减轻,MDA生成明显减少,心肌细胞凋亡率明显降低,Bcl-2表达增加,Bax表达减少,caspase-3和caspase-9阳性细胞数明显减少。结论丙泊酚减轻I-R所致的心肌线粒体过氧化损伤,抑制线粒体途径的细胞凋亡,可能是其心肌保护作用机制之     

丙戊酸钠诱导K562细胞凋亡的机制探讨

目的 探讨丙戊酸钠(VPA)诱导K562细胞凋亡的可能机制.方法 将K562细胞分为经VPA 2.0mmol/L处理的实验组(A组)和正常对照组(B组),分别培养24、48、72 h.流式细胞术检测细胞凋亡率和线粒体膜电位改变,分光光度法检测半胱氨酸天门冬氨酸蛋白酶(Caspase)8、Caspase-9蛋白活性.结果 A组细胞凋亡率、细胞线粒体跨膜电位破坏率呈时问依赖性地增加,且明显高于B组(P<0.05);与B组相比,A组不同时间的Caspase-8、Caspase-9活性均上调(P<0.05).结论 VPA诱导K562细胞凋亡的机制可能与线粒体跨膜电位崩溃或死亡途径有关.     

三氧化二砷诱导类风湿性关节炎滑膜细胞凋亡机制的体外研究

目的:探讨三氧化二砷(As2O3)诱导人成纤维样滑膜细胞( HFLS) 凋亡的可能机制.方法:用As2O3作用于类风湿性关节炎(RA)患者HFLS后,酶联免疫吸附试验(ELISA)检测凋亡过程中Caspase-3水平,逆转录聚合酶链反应(RT-PCR)检测Caspase-3和Bcl-2 mRNA表达.结果:凋亡早期细胞色素C(CytC)水平升高,不同浓度As2O3作用HFLS后不同时间胞浆中CytC含量差别有统计学意义(F浓度=2 541.065,F时间 =4 433.196,P < 0.01),且时间和浓度间存在着交互效应(F交互=825.472,P < 0.01).不同浓度的As2O3作用后细胞中Caspase-3 mRNA表达显著增强, Bcl-2 mRNA表达显著减弱,且分别与As2O3浓度呈正相关和负相关.结论:As2O3可能通过升高CytC及Caspase-3,抑制Bcl-2诱导HFLS凋亡.     

manumycin诱导舌鳞癌Tca8113细胞凋亡的作用及机制

目的研究manumycin抑制Tca8113细胞的生长和诱导细胞凋亡的作用及机制。方法细胞毒作用以MTT法测定;凋亡细胞形态以Hoechst33258染色后荧光显微镜观察;凋亡率的检测以AnnexinV染色,流式细胞仪检测;细胞内活性氧(ROS)和线粒体跨膜电位(ΔΨm)分别用DCFH-DA和DiOC6荧光探针标记,流式细胞仪检测;蛋白质定量检测以Westernblot法。结果manumycin浓度依赖性抑制Tca8113细胞的生长,IC50为(11.33±0.63)μmol.L-1;manumycin可浓度和时间依赖性诱导Tca8113细胞的凋亡,过氧化物清除剂N-乙酰半胱氨酸能清除manumycin介导的细胞活性氧的增加,抑制线粒体跨膜电位降低及细胞凋亡。结论manumycin体外显著抑制舌鳞癌Tca8113的生长及诱导细胞凋亡,其机制可能通过激活线粒体依赖性凋亡通路有关。     

三氧化二砷诱导肿瘤细胞凋亡途径的研究

目的　探讨细胞线粒体跨膜电位 (Δψm)和半胱氨酶3(Caspase 3)在三氧化二砷 (As2 O3 )诱导肿瘤细胞凋亡过程中的作用。方法　以Namalwa、SGC790 1和Bcap37细胞为体外模型 ,流式细胞仪检测亚G1期细胞含量和细胞膜磷脂酰丝氨酸 (PS)外翻量等方法鉴定细胞凋亡 ;碘化丙啶 (PI) /Rhodamine(Rh12 3)双染色检测Δψm变化并观察了Caspase 3抑制剂DEVD CHO对As2 O3 诱导肿瘤细胞凋亡的影响。结果　As2 O3 诱导肿瘤细胞凋亡效应与其诱导细胞Δψm下降和Caspase 3活性升高相关 ,抑制Caspase 3活性后As2 O3 使细胞选择坏死通路。结论　As2 O3 可能通过诱导细胞Δψm下降激活Caspase 3并最终使肿瘤细胞凋亡。     

Caspases家族成员促进DADAG诱导的人白血病HL—60细胞凋亡

目的:研究caspases家族成员在二乙酰二脱水卫矛醇(DADAG)诱导人白血病HL-60细胞凋亡中的作用.方法:MTT法观察DADAG的体外抗增殖作用;透射电镜、DNA梯形条带和流式细胞仪检测HL-60细胞凋亡;caspase-3检测试剂盒和Western blot法分析caspases家族成员.结果:DADAG明显抑制HL-60细胞增殖和诱导细胞凋亡.DADAG处理HL-60细胞24h后,caspase-3酶活性达峰值,同时聚腺苷二磷酸核糖聚合酶(PARP)、lamin B和DFF45蛋白开始出现断裂片段.Caspase-3抑制剂z-DEVD·fmk可部分逆转DADAG诱导的HL-60细胞凋亡,而caspases广谱抑制剂z-VAD·fmk可完全逆转此作用.结论:Caspases在DADAG诱导HL-60细胞凋亡中起重要作用,它们通过酶解底物PARP、DFF45和lamin B促进细胞凋亡.     

Study on preliminary mechanism of apoptosis in HepG-2 by CSA

Objective To study on the mechanism of killing and apoptosis inducing effect of total alkaloid in the CSA(Capparis spinosa L.alkaloid,CSA)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSA on human hepatocarcinoma cell Line HepG-2 was measured by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSA on the HepG-2 cells were measured by flow cytometry.In addition,effect of intracellular Ca2+ level of the CSA on the HepG-2 cells was studied by laser confocal microscope.Results The CSA has obvious cytotoxicity on the HepG-2 and seems to be dose-dependent,and its IC50 value is 162.4 μg·mL-1.The HepG-2 cells have characteristic morphologic changes of apoptosis by the function of CSA,and the apoptosis percentage is higher than the natural one.The progress of cells cycle from S phase to G2 phase has been blocked,and the mitochondria membrane potential is markedly decreased,and the intracellular Ca2+ level is increased by the function of CSA.Conclusions The CSA has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

Effect on apoptosis、mitochondrial membrane potential and Ca~(2+) concentration in HepG-2 by CSEO

Objective To study on the mechanism of growth inhibiting and apoptosis inducing effect of total alkaloid in the CSEO(Capparis spinosa L.essential oil,CSEO)on human hepatocarcinoma cell Line HepG-2.Methods The growth inhibiting effect of the CSEO on human hepatocarcinoma cell Line HepG-2 was measured by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.The changing of mitochondrion membrane potential induced by CSEO was observed by staining with Rhodamine123.Effect of the CSEO on intracellular Ca2+ level of the HepG-2 cells was measured by laser confocal microscope.Results The CESO has obvious growth inhibiting effect on the HepG-2 and seems to be dose-dependent,and its IC50 is 127.5 μg·mL-1.The characteristic apoptosis morpha of HepG-2 cells has been observed,and the apoptosis percentage increase to 44.447% in the 300 μg·mL-1 dosage group.In addition,the progress of cells cycle of G1 period has been blocked,and the cellular proportion in S and G2 period is decreased in the 75 μg·mL-1 and 150 μg·mL-1 dosage groups by the function of CSEO for 48 h.The mitochondria membrane potential(Δψm)effected by CESO is decreased,while the curve moves toward left.In addition,the intracellular Ca2+ level is increased by the function of CESO in the middle and high dose groups.Conclusions The CESO has obviously growth inhibiting and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

大花紫玉盘素诱导肿瘤多药抗药性细胞凋亡及其机制

目的比较研究番荔枝内酯大花紫玉盘素(uvarigrin)诱导多药抗药性KBv200细胞及其亲本KB细胞凋亡及其机制。方法以MTT法进行细胞毒测定；用Annexin V FITC染色及流式细胞仪检测细胞凋亡。活性氧(ROS)测定以DCFH-DA标记，细胞线粒体跨膜电位(ΔΨ_m)测定用DiOC₆标记，均以流式细胞仪检测。Caspase-9激活的测定用Western blotting法。结果大花紫玉盘素对KBv200细胞及其亲本KB细胞的生长均有明显的抑制作用；大花紫玉盘素不仅能介导KB细胞凋亡，而且也能介导KBv200细胞凋亡；大花紫玉盘素作用于KBv200细胞及其亲本KB细胞12，24和48 h，均引起ROS升高以及ΔΨm降低，而且呈时间依赖性。Western blotting方法分析显示Caspase-9被激活。结论大花紫玉盘素可能通过线粒体通路诱导细胞凋亡。     

莨菪亭对PC3细胞增殖和凋亡的影响

目的:研究莨菪亭对人前列腺癌细胞PC_3增殖的作用和莨菪亭是否能引起PC_3细胞的凋亡。方法:用细胞生长曲线,MTT试验和酸性磷酸酶(ACP)活性来测定细胞增殖,考马斯亮蓝法测细胞内蛋白质的含量,光镜、透射电镜和荧光显微镜观察莨菪亭引起的形态学变化。用荧光显微镜和流式细胞仪确定凋亡率和细胞的周期分布。结果:莨菪亭对PC_3,PAA和Hela细胞的IC_(50)分别为(157±25),(154±51)和(294±100)mg/L,莨菪亭时间和浓度依赖性地抑制PC_3细胞的增殖,并引起细胞内蛋白质含量减少和ACP活性降低。经莨菪亭处理后,在光镜、透射电镜和荧光显微镜下可观察到莨菪亭引起的典型的凋亡形态学变化,流式细胞仪测定显示经莨菪亭0,100,200和400mg/L处理后PC_3细胞的凋亡率分别为0.3％,2.1％,9.3％和35％,G_2期细胞显著减少。结论:莨菪亭抑制PC_3细胞增殖且可引起PC_3细胞的凋亡。     

Effects of GM-CSF, IL-3, and GM-CSG/IL-3 fusion protein on apoptosis of human myeloid leukemic cell line Tf-1 induced by irradiation

AIM: To observe the effects of three cytokines on the apoptosis of Tf-1 cells induced by y irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry, and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf-1 cells were irradiated by y     

金喜素抑制JM细胞增殖并诱导其凋亡的流式细胞术分析

金喜素(topotecan,TPT)为喜树碱类化疗药物,可以选择性地抑制拓扑异构酶I(Topoisomerase I,Topo I)[1].以往的研究表明,该药物可以用于慢性粒单细胞白血病、骨髓增生异常综合征(MDS)和实体瘤[2～4]等的治疗.T淋巴细胞白血病在临床上多见于儿童,起病急,病情重,严重危害患儿的健康.JM为急性T淋巴母细胞白血病细胞株[5].金喜素对JM细胞株作用的研究未见文献报道.本研究旨在观察金喜素对JM细胞株增殖的抑制作用及其诱导该细胞株发生凋亡的作用.     

右美托咪定对布比卡因所致神经细胞毒性的保护作用

摘要： 目的 探讨右美托咪定对布比卡因所致神经细胞毒性的保护作用。方法 体外培养小鼠神经母细胞瘤细胞株 N2a 细胞, 取对数期细胞分为 4 组： 对照组细胞培养液不加任何药物; 布比卡因组细胞中加入 1 000 µmol/L 布比卡因; 50、 200 µmol/L 右美托咪定浓度组细胞中加入 1 000 µmol/L 布比卡因后, 再分别加入 50、 200 µmol/L 右美托咪定。各组细胞加入药物后继续培养 24 h, 以 MTT 法检测细胞存活率; 流式细胞术检测细胞凋亡、 活性氧 （ROS）水平、 线粒体膜电位及 Caspase-3 的表达。结果 在 1 000 µmol/L 布比卡因作用下, 各药物组 N2a 细胞的存活率明显降低, 同时线粒体膜电位显著下降, 而细胞的凋亡率、 胞内 ROS 水平和 Caspase-3 表达则显著升高; 50、 200 µmol/L 右美托咪定可抑制布比卡因引起的 N2a 细胞毒性, 使细胞的存活率及线粒体膜电位均明显升高, 同时降低细胞的凋亡率、 胞内 ROS 水平和 Caspase-3 表达, 200 µmol/L 右美托咪定的变化较 50 µmol/L 更为明显。结论 右美托咪定可减轻布比卡因对 N2a 细胞的毒性作用, 其可能是通过抑制 ROS 的生成、 改变线粒体膜电位、 降低 Caspase-3 的表达, 从而抑制细胞的凋亡来实现的。