干扰素对U937细胞的诱导凋亡作用及其作用机制

目的 探讨干扰素α对白血病U937细胞的生长抑制作用及其作用机制。方法 以不同浓度的干扰素α作用于体外培养的U937细胞，MTT法检测细胞生长抑制率，应用流式细胞仪及hoechst33258荧光染色法观察细胞凋亡，TRAP-PCR—ELISA法检测细胞凋亡前后的端粒酶活性。结果 干扰素α可显的降低U937细胞端粒酶活性，抑制细胞的生长及诱导细胞发生凋亡。并呈现出明显的量-效与时-效关系。结论 干扰素α能抑制U937细胞的生长并诱导细胞发生凋亡，降低细胞端粒酶活性可能是其重要作用机制之一。     

磷酸氯喹诱导U937细胞凋亡机制的研究

目的 将磷酸氯喹作用于白血病细胞株U937,观察其对白血病细胞凋亡的影响;并研究磷酸氯喹是否通过逆转凋亡相关基因PNAS-2蛋白在白血病细胞中异常的亚细胞定位,从而促进细胞凋亡的机制。方法 将不同剂量的磷酸氯喹加入体外培养至对数生长期的白血病细胞株U937培养液中。MTT法检测细胞增殖;流式细胞仪和激光共聚焦显微镜检测细胞凋亡,同时观察磷酸氯喹对U937细胞中PNAS-2蛋白亚细胞定位的影响。结果 50 μg /mL磷酸氯喹可显著促进U937细胞发生凋亡,并使白血病细胞中PNAS-2蛋白的异常亚细胞定位发生改变。结论 磷酸氯喹对白血病细胞株U937有促进凋亡的作用,其凋亡机制可能是使PNAS-2蛋白在白血病细胞中异常的亚细胞定位恢复正常,对于临床治疗白血病的具有潜在的应用价值。     

ABT-737对人急性髓细胞白血病细胞U937诱导凋亡作用及机制

目的探讨Bcl-2小分子抑制剂ABT-737对人急性髓细胞白血病细胞U937诱导凋亡作用及机制。方法以人急性髓细胞白血病细胞U937为研究对象,观察不同浓度梯度(0、0.625、1.25、2.5、5.0、10μmol/L)ABT-737对U937细胞增殖抑制及诱导凋亡作用,并检测ABT-737作用前后Bcl-2 mRNA水平和蛋白表达水平改变。CCK-8检测细胞生长抑制率,Annexiin-V/PI双染检测细胞凋亡率,RT-PCR方法检测Bcl-2 mRNA水平,Western blot检测Bcl-2蛋白表达。结果 ABT-737可显著抑制U937细胞生长,随着ABT-737浓度(0、0.625、1.25、2.5、5.0、10μmol/L)的增加,U937细胞数量逐渐减少。其中2.5μmol/L ABT-737即可使U937细胞出现明显增殖抑制,作用24 h后其存活率与对照组(0μmol/L)比较差异有统计学意义(P<0.05)。ABT-737可显著诱导U937细胞凋亡,随着ABT-737浓度(0、0.625、1.25、2.5、5.0、10μmol/L)的增加,凋亡率逐渐增加。其...     

蟾蜍灵对白血病U937细胞生长抑制和凋亡诱导作用

蟾蜍灵是蟾蜍毒素的主要成分之一,蟾蜍毒素具有强心、麻醉、解毒、止痛、开窍、醒神等药理作用,已被广泛应用于临床。近年来发现,含有蟾蜍毒素的中药制剂在体外能抑制多种肿瘤细胞生长,诱导白血病细胞的分化。临床观察提示,含有蟾蜍毒素的中药制剂对肝癌、肺癌、胃癌、食管癌、白血病等具有明显的抑制作用。Numazawa[1]研究表明,蟾蜍毒素的各种成分以剂量依赖的方式抑制K562细胞生长。在所有的蟾蜍毒素中,蟾蜍灵是诱导白血病细胞分化最有效的成分,蟾蜍灵在较低浓度(1×10-8~1×10-9mol/L)能够在较宽的范围内(有较宽的白血病细胞谱)引起人…     

磷酸氯喹诱导U937细胞凋亡机制的研究

目的 将磷酸氯喹作用于白血病细胞株U937,观察其对白血病细胞凋亡的影响;并研究磷酸氯喹是否通过逆转凋亡相关基因PNAS-2蛋白在白血病细胞中异常的亚细胞定位,从而促进细胞凋亡的机制.方法 将不同剂量的磷酸氯喹加入体外培养至对数生长期的白血病细胞株U937培养液中.MTT法检测细胞增殖;流式细胞仪和激光共聚焦显微镜检测细胞凋亡,同时观察磷酸氯喹对U937细胞中PNAS-2蛋白亚细胞定位的影响.结果 50 μg/mL磷酸氯喹可显著促进U937细胞发生凋亡,并使白血病细胞中PNAS-2蛋白的异常亚细胞定位发生改变.结论 磷酸氯喹对白血病细胞株U937有促进凋亡的作用,其凋亡机制可能是使PNAS-2蛋白在白血病细胞中异常的亚细胞定位恢复正常,对于临床治疗白血病的具有潜在的应用价值.     

EGCG通过下调Bcl-2诱导KG1A白血病细胞凋亡

目的研究EGCG对KG1A白血病细胞的作用,探讨其作用机制。方法使用人的白血病细胞株KG1A细胞。MTT细胞毒性测定评价对白血病细胞的作用,使用annexin v染料标记细胞,应用流式细胞仪检测细胞凋亡。RT-PCR检测KG1A细胞Bcl-2家族mRNA的表达。结果EGCG引起KG1A细胞活性的下降。EGCG诱导KG1A细胞凋亡,且呈时间依赖性。EGCG诱导凋亡与下调Bcl-2和Bcl-xl有关。结论EGCG可以通过下调Bcl-2和BCl-xl诱导KG1A细胞凋亡。这些结果有助于提供新的有效联合方案,尤其是针对复发难治白血病的治疗。     

EGCG通过下调Bcl-2诱导KG1A白血病细胞凋亡宰

目的 研究EGCG对KGIA白血病细胞的作用,探讨其作用机制.方法 使用人的白血病细胞株KG1A细胞.MTT细胞毒性测定评价对白血病细胞的作用,使用annexin v染料标记细胞,应用流式细胞仪检测细胞凋亡.RT-PCR检测KGIA细胞Bcl-2家族mRNA的表达.结果 EGCG引起KG1A细胞活性的下降.ECA2G诱导KG1A细胞凋亡.且呈时间依赖性.EGCG诱导凋亡与下调Bcl-2和Bcl-xl有关.结论 EGCG可以通过下调Bcl-2和Bcl-xl诱导KG1A细胞凋亡.这些结果有助于提供新的有效联合方案,尤其是针对复发难治白血病的治疗.     

抗PNAS-2单链抗体对白血病U937细胞凋亡的影响

目的观察抗PNAS-2单链抗体与PNAS-2抗原特异性结合的能力,并在此基础上探究其对急性单核细胞白血病U937细胞凋亡的影响。方法运用反转录病毒转染法将抗PNAS-2单链抗体表达载体pMIG-PNAS-2ScFv转入U937细胞(pMIG-PNAS-2ScFv组),同时设转染pMIG空载体(NC组)及未转染载体的U937细胞(U937组)为对照。流式细胞仪检测转染效率;激光共聚焦显微镜观察单链抗体表达情况及与靶抗原特异性结合的能力;流式细胞仪和激光共聚焦显微镜检测细胞凋亡;Western blotting观察PNAS-2表达及促凋亡因子caspase-3激活情况。结果成功将抗PNAS-2单链抗体编码序列转入U937细胞,所表达的单链抗体能与PNAS-2抗原特异性结合。与NC组和U937组相比,pMIG-PNAS-2ScFv组细胞凋亡率明显上升(P<0.05),活化型caspase-3表达增加,但PNAS-2含量并无变化。结论抗PNAS-2单链抗体能有效结合U937细胞内的PNAS-2抗原并有促进U937细胞凋亡的作用,其促凋亡机制可能与PNAS-2功能位点被封闭后功能失活而无法发挥抑凋亡作用有关。     

刺梨汁促人急性单核细胞白血病U937细胞凋亡作用的研究

目的：探讨刺梨汁（RRTJ）促U937细胞凋亡作用和可能的机制。方法：不同浓度RRTJ在体外作用于人急性单核细胞白血病U937细胞株。应用光学显微镜观察细胞形态变化；细胞计数测定细胞生长情况；流式细胞仪分析细胞生长周期的变化；逆转录聚合酶链反应法（RT—PCR）观察凋亡相关基因bcl-2、bax表达的变化；硝酸还原酶法测定细胞一氧化氮（NO）分泌量；并通过测定细胞上清液乳酸脱氢酶（LDH）活性分析RRTJ对细胞的毒性作用。结果：刺梨汁显著抑制U937细胞生长，并表现出剂量依赖性。随RRTJ浓度增加，U937细胞凋亡率增高，baxmRNA表达量上升，而bcl-2mRNA表达水平明显下降。U937细胞NO释放量也随RRTJ浓度增加而增加。结论：RRTJ可能是通过影响细胞生长周期、降低bcl-2／bax比值，同时诱导NO分泌，促进U937细胞凋亡。     

顺铂增强抗人DR5单克隆抗体对白血病U937细胞的凋亡作用

通过探讨鼠抗人DR5单克隆抗体(mDRA-6)与顺铂(DDP)联合应用对白血病细胞U937凋亡作用的影响,得出结论:抗人DR5单克隆抗体mDRA-6与DDP对U937细胞具有显著的协同杀伤作用.     

活性氧、白细胞和精子凋亡关系的探讨

目的 探讨活性氧与精子凋亡的相关性及其影响.方法 分析21例男性不育患者样本,活性氧含量采用分光光度比色法检测,瑞-姬染色检测精子凋亡率,采用Percoll法进行精子优选.结果 本底水平活性氧含量、白细胞受激发后产生活性氧含量、白细胞和精子受激发后产生活性氧含量与精子凋亡率均有显著正相关性(P＜0.05,P＜0.01);Percoll优选后,加入H2O2组精子凋亡率显著高于对照组(P＜0.001),且0.2 mM过氧化氢组精子凋亡率均高于相应的0.02 mM过氧化氢组(P＜0.05).结论 精子凋亡率与ROS浓度有正相关关系,外加H2O2增高,精子凋亡率增高.     

2型糖尿病大鼠肾脏活性氧物质与NO关系的探讨

目的研究糖尿病大鼠肾脏活性氧物质与一氧化氮(NO)系统的关系。方法制备高脂高糖加小剂量链脲佐菌素(35mg/kg)诱导的2型糖尿病大鼠模型。将大鼠分为正常对照组10只和糖尿病组20只;观察糖尿病大鼠肾脏氧化应激状态及NO系统的表达,肾功能改变及抗氧化指标与NO的相关性分析。结果糖尿病大鼠成型12周时SOD,SOD/MDA降低,MDA升高,与正常对照组比较差异有统计学意义;NO、总NOS、iNOS、cNOS均较正常对照组明显升高。肾脏肥大指数、尿微量清蛋白、基质比、肾小球截面积与SOD/MDA呈负相关,与NO、总NOS呈正相关;SOD/MDA与总NOS、NO之间存在负相关。结论氧化应激,NO均参与糖尿病肾病的发生、发展,它们之间的平衡可能是抗氧化治疗在糖尿病肾病中的关键。     

Role of reactive oxygen species in triptolide-induced apoptosis of renal tubular cells and renal injury in rats

This study investigated the role of reactive oxygen species(ROS) in the pathogenesis of triptolide-induced renal injury in vivo.Rats were randomly divided into 4 groups(n=5 in each):triptolide group in which the rats were intraperitoneally injected with triptolide solution at a dose of 1 mg/kg of body weight on day 8;control group in which the rats received a single intraperitoneal injection of 0.9% physiological saline on day 8;vitamin C group in which the rats were pretreated with vitamin C by gavage at a dose of 250 mg/kg of body weight per day for 7 days before the same treatment as the control group on day 8;triptolide+vitamin C group in which the rats were first subjected to an oral administration of vitamin C at a dose of 250 mg/kg of body weight per day for 7 days,and then to the same treatment as the triptolide group on day 8.All the rats were sacrificed on day 10.Blood samples were collected for detection of plasma creatinine(Pcr) and plasma urea nitrogen(PUN) concentrations.Both kidneys were removed.The histological changes were measured by haematoxylin-eosin(HE) staining.The production of ROS was determined by detecting the fluorescent intensity of the oxida-tion-sensitive probe rhodamine 123 in renal tissue.Renal malondialdehyde(MDA) content was meas-ured to evaluate lipid peroxidation level in renal tissue.TUNEL staining was performed to assess apop-tosis of renal tubular cells.Renal expression of apoptosis-related proteins Bcl-2,Bax,Bid,Bad,Fas and FasL,as well as corresponding encoding genes were assessed by Western Blotting and real-time PCR.The results showed that triptolide treatment promoted the generation of a great amount of ROS,up-regulated the expression of Bax,Bid,Bad,Fas and FasL at both protein and mRNA levels,as well as the ratio of Bax to Bcl-2,and caused the apoptosis of renal tubular cells and renal injury.However,pretreatment with an antioxidant,vitamin C,significantly reduced the generation of ROS and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury.It was concluded that ROS plays a critical role in triptolide-induced apoptosis of renal tubular cells and renal injury.The protective administration of vitamin C may help alleviate triptolide-induced renal injury and nephrotoxicity.     

活性氧在梗阻性黄疸大鼠肝细胞凋亡中的作用研究

目的 探讨活性氧在梗阻性黄疸大鼠肝细胞凋亡中的作用.方法 测定利用体外原代培养的大鼠肝细胞和大鼠梗阻性黄疸模型中还原型谷胱甘肽、活性氧、谷胱甘肽过氧化物酶活性、活性氧以及肝细胞凋亡指数等指标变化,并行统计学分析.结果 体外实验中肝细胞与终浓度为甘氨鹅脱氧胆酸钠(GCDC)后培养2h和4h后细胞凋亡率达(43.50±7.09)%和(53.38±5.24)%,其内源性还原型谷胱甘肽消耗呈时间依赖性.梗阻性黄疸模型建立后,随着胆盐水平升高,单纯梗阻性黄疸组大鼠肝组织丙二醛水平上升,还原型谷胱甘肽、谷胱甘肽过氧化物酶活性进行性下降.Tunel染色发现大鼠肝组织细胞出现凋亡,并随时间延长而增多.亚硒酸钠治疗组上述指标变化趋势与单纯梗阻性黄疸组相似,但其各时间点丙二醛水平和凋亡率显著低于单纯梗阻性黄疸组,而还原型谷胱甘肽水平和谷胱甘肽过氧化物酶活性则明显高于后者.结论 ROS在肝脏功能损害中起着重要的作用.亚硒酸钠是一种颇有应用潜能的肝细胞凋亡拮抗剂.     

活性氧激活线粒体凋亡通路是亚硒酸钠诱导NB4细胞凋亡的重要机制

目的 探讨亚硒酸钠诱导NB4细胞凋亡过程中,活性氧引起的线粒体膜电位丧失及凋亡作用的相关机制。方法 用活性氧荧光探针DCFH-DA探测经亚硒酸钠诱导的NB4细胞内活性氧的含量。流式细胞术检测细胞凋亡率及线粒体膜电位变化。Westem blot检测胞浆中细胞色素C含量的变化及细胞总蛋白中Bcl-xl、Bax和Bid的含量变化及切割行为。同时应用活性氧清除剂MnTmPy及活性氧激活剂BSO预处理细胞,观察Bid及凋亡的变化。结果 活性氧清除剂MnTmPy可有效抑制亚硒酸钠诱导的NIM细胞凋亡、线粒体膜电位丧失及Bid的切割;而BSO可促进线粒体膜电位丧失。结论 亚硒酸钠诱导NIM细胞凋亡的可能机制是通过刺激细胞产生活性氧,一方面下调抗凋亡蛋白Bcl-xl,另一方面激活线粒体打孔蛋白Bax和Bid,损伤线粒体使膜电位降低,促使线粒体释放细胞色素C。     

低糖诱导人脐静脉内皮细胞一氧化氮与活性氧变化的研究

目的观察低浓度葡萄糖对人脐静脉内皮细胞一氧化氮(NO)与活性氧(ROS)变化的影响。方法以体外培养人脐静脉内皮细胞株HUVEC-12为研究对象,将实验分为正常对照组(5.5mmol/L葡萄糖)、低糖组(2.8mmol/L葡萄糖)、无糖组(0mmol/L葡萄糖)。分别用2.8、0mmol/L葡萄糖干预HUVEC-12细胞,经过1、2、4、12h后,硝酸还原酶法测定NO产量,化学比色法测定一氧化氮合成酶(NOS)活性,Dihydroethidium荧光探针法测定细胞内ROS水平。结果与正常对照组相比,低糖组与无糖组NO水平降低(P<0.01),NOS活性下降(P<0.01),细胞内ROS水平升高(P<0.01),均呈剂量依赖关系。同一浓度组内随着时间的延长,内皮细胞NO水平、NOS活性进一步降低(P<0.01),ROS水平进一步升高(P<0.05),各指标都具有浓度和时间依赖性。结论低糖可导致内皮细胞功能障碍,NO水平降低,NOS活性下降,其机制可能与低糖导致内皮细胞氧化应激损伤有关。     

氢气饱和生理盐水对高糖所致内皮细胞内活性氧生成和凋亡的影响

目的探讨氢气(H2)饱和生理盐水对高糖所致内皮细胞内活性氧(ROS)生成和凋亡的影响。方法细胞分为对照组、高糖组、高糖+H2饱和生理盐水组(H2组)3组,流式细胞仪检测细胞内ROS,Realtime PCP检测氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)mRNA,细胞凋亡ELISA试剂盒检测细胞凋亡。结果 H2组内皮细胞内ROS较高糖组明显减少(2.75±0.37vs 6.35±1.29,P<0.01),而SOD和GSH-Px mRNA水平明显增高(SOD:0.74±0.06vs 0.26±0.03,P<0.01;GSH-Px:0.68±0.05vs 0.31±0.04,P<0.01),细胞凋亡水平明显降低(0.38±0.02vs 0.89±0.05,P<0.01)。结论 H2饱和生理盐水可减少高糖诱导的内皮细胞内ROS增多和抗氧化酶的消耗,并减少高糖诱导的细胞凋亡。     

高糖对人腹膜间皮细胞生成活性氧的影响

[目的]研究不同浓度的葡萄糖对人腹膜间皮细胞（human peritoneal mesothelial cells,HPMCs）生成活性氧的影响。[方法]HPMCs体外培养并进行免疫组化鉴定,取第3代细胞用于实验。分别按照不同的实验要求将细胞分组。不同葡萄糖浓度干预组：①空白对照组;②1．5％葡萄糖组;③2．5％葡萄糖组;④4．25％葡萄糖组。不同作用时间组：①空白对照组;②2．5％葡萄糖组。两组细胞分别培养12、24、48h。应用流式细胞仪检测细胞对氧化剂敏感的2．7-二氢二氯荧光素（2’,7’-dichlorodihydrofluorescein,DCFH）发生氧化所产生的荧光量,从而间接的反映细胞内活性氧（reactive oxygen species,ROS）的水平,观察随葡萄糖浓度变化及不同作用时间对人腹膜间皮细胞产生活性氧的影响。[结果]（1）与空白对照组比较,高糖干预人腹膜间皮细胞后,其细胞内平均荧光强度明显升高（P〈0．05）,并且随葡萄糖浓度升高而逐渐增高,其中4．25％葡萄糖组最高。（2）与空白对照组比较,随着时间的延长,高糖作用下细胞内平均荧光强度明显升高（P〈0．05）,培养48h其水平最高。[结论]高糖可刺激体外培养的人腹膜间皮细胞ROS的生成。     

Apoptosis susceptibility of tumor cells to arsenic trioxide and the inherent cellular level of reactive oxygen species

Objective To explore the association of in herent cellular reactive oxygen species (ROS) levels with susceptibility of the tumor cells to apoptosis induction by arsenic trioxide (As(2)O(3)).Methods Low concentration (2μmol/L) of As(2)O(3) was administered to two cultured leukemic cell lines, NB4 and U937, and two esophageal carcinoma cell lines, EC1 .71 (also named EC/CUHK1) and EC1867, to confirm the difference in apoptosis su sceptibility of NB4 versus U937 and of EC1.71 versus EC1867. Dihydro genrhodamine 123 (DHR123), used as a ROS capture agent, was incubated with cell s in the absence of As(2)O(3). Fluorescence intensity of rhodamine 123, the pro duct of cellular oxidation of DHR123, was detected by flow cytometry and ROS was measured. Results Low concentration of As(2)O(3) induced apoptosis was more likely to occur in NB 4 and EC1.71 cells than in U937 and EC1867 cells, or NB4 was more sensitive tha n U937, and EC1.71 more sensitive than EC1867 to As(2)O(3). The inherent cellu lar ROS level is higher in NB4 than in U937, and also higher in EC1.71 than in EC1867 . Conclusions The difference in cellular ROS level is positively associated with cellular susc eptibility to apoptotis induction by As(2)O(3). The inherent ROS level might be important in defining apoptotic susceptibility to As(2)O(3).     

Mechanisms of MPP+-induced PC12 cell apoptosis via reactive oxygen species

Apoptosis of dopaminergic neurons in the nigrostriatal projection plays a crucial role in the pathogenesis of Parkinson’s disease (PD). Although the detailed mechanisms responsible for dopaminergic neuron loss are still under investigation, oxidative stress is identified as a major contributor for neuronal apoptosis. In the current study, we studied the effects of MPP+, a substrate that mimics oxidative stress, on neuron-like PC12 cells and the underlying mechanisms. PC12 cells were cultured and treated by 100 μmol/L MPP+ for 4, 8, 16, 24 and 48 h, respectively. For drug pretreatment, the PC12 cells were incubated with N-acetyl-l-cysteine (NAC, 5 mmol/L), an antioxidant, SP600125 (20 μmol/L) or PD98059 (100 μmol/L), two pharmacological inhibitors of JNK and ERK1/2, for 1 h before addition of MPP+. Cell apoptosis was measured by flow cytometry. The mRNA expression of Cu2+/Zn2+-SOD, GSH-Px, Bcl-2 and Bax was detected by RT-PCR. The protein expression of p-ERK1/2 and p-JNK was determined by Western blotting. Our results showed that MPP+ exposure could induce substantial PC12 cell apoptosis. The pretreatment of SP600125 or PD98059 could effectively reduce the apoptosis rate by reducing the ratio of Bax/Bcl-2 mRNA levels. MPP+ exposure also induced high level of reactive oxy-gen species (ROS), marked by dramatic increase of Cu2+/Zn2+-SOD and GSH-Px mRNA levels. The elevated ROS was strongly associated with the activation of JNK and ERK1/2 signal pathways after MPP+ exposure, since the pretreatment of NAC significantly reduced the upregulation of p-JNK and p-ERK1/2. Finally, the pretreatment of SP600125, but not PD98059, alleviated the increase of Cu2+/Zn2+-SOD and GSH-Px mRNAs induced by MPP+, suggesting that the activation of the JNK signal pathway, but not the ERK1/2 signal pathway, could, in some degree, antagonize the generation of ROS induced by oxidative stress. In conclusion, our results suggest that JNK and ERK1/2 signal pathways, which are activated via ROS, play a crucial role in neuronal apoptosis ind