辛伐他汀联合阿糖胞苷对K562细胞增殖与凋亡的影响

目的 探讨辛伐他汀(SV)联合阿糖胞苷(Ara-C)对K562细胞增殖与凋亡的影响.方法 不同浓度SV和Ara-C单用或者联合处理K562细胞,对照组为K562细胞.药物作用24、48、72 h后收集细胞,分别观察各组细胞形态,采用MTT法检测不同组别细胞的生长抑制率,采用流式细胞术检测细胞早期凋亡率、细胞坏死比例.结果 SV联合Ara-C组与单药组相比细胞形态明显有核固缩现象,且可见凋亡小体形成,并且随着处理时间的增加,抑制率也增大.其中15 μmol/L SV联合20 μmol/LAra-C的细胞抑制作用最为显著,72 h细胞抑制率为(72±1)%,明显高于15 μmol/L SV组的(45±2)%和20μmol/LAra-C组的(44±0)%(P＜0.01),表现为协同抑制作用(24、48 h金氏Q值为1.24和1.19).流式细胞术检测发现20、15和10μmol/LSV组K562细胞早期凋亡率AnnexinV明显高于对照K562细胞(P＜0.01),而且随着时间延长和剂量的增大早期凋亡率也增加(P＜0.05).20和15 μmol/LSV组早期凋亡率均高于10 μmol/LSV组,而前两者之间差异无统计学意义(P＞0.05).晚期凋亡细胞率(PI)各组中差异均无统计学意义(P＞0.05).结论 SV体外抑制K562细胞增殖及诱导细胞凋亡,SV与Ara-C具有协同作用,增加了K562细胞对化疗药物的敏感性.15 μmol/L可能为SV体外最佳作用浓度.     

葛根总黄酮诱导白血病细胞株凋亡及其分子机制的研究

目的 探讨葛根总黄酮(PR)对慢性粒细胞白血病(CML)细胞株K562和急性早幼粒细胞白血病(APL)细胞株NB4细胞增殖及凋亡的影响.方法 采用MTT法检测PR对K562细胞、NB4细胞的增殖抑制率;光学显微镜及荧光显微镜观察细胞形态改变;Hoechest33258荧光染色AnnexinV/PI双染法检测细胞凋亡率;DNA PI染色法分析细胞周期及亚二倍体峰.Western blot分别检测NB4细胞JNK、PARP、bcl-2、Caspase3,K562细胞bcr-abl、p53、bcl-2、Fas/FasL蛋白表达的变化.结果 12.5～200 μg/ml PR均能抑制K562、NB4细胞增殖.光学显微镜及荧光显微镜下观察到核固缩、凋亡小体等典型的细胞凋亡改变;Annexin V+/PI-细胞呈时间-剂量依赖性增加;DNA PI染色法发现细胞亚二倍体比例增加,G1期比例下降、S期比例增加.PR呈时间-剂量依赖性抑制K562细胞、NB4细胞增殖,诱导细胞凋亡.不同浓度PR干预后K562细胞bcr-abl蛋白水平呈浓度依赖性下调(F=18.74,P<0.05),而bcl-2则无明显变化;p53表达呈浓度依赖性上调;Fas/FasL表达无明显变化.NB4细胞JNK、PARP及Caspase 3蛋白表达与PR浓度呈正相关,与凋亡抑制蛋白bcl-2则呈负相关(F=42.32,P<0.05).结论 PR能有效抑制K562、NB4细胞增殖,阻滞细胞周期进程,诱导细胞凋亡,但分子机制不同.提示一定浓度PR具有较广谱的抗白血病效应.     

小干扰RNA逆转三氧化二砷耐药的白血病K562/AS2细胞Topo Ⅱ基因表达的实验研究

目的 研究小干扰RNA片段(shRNA)对三氧化二砷(ATO)耐药的白血病细胞株K562/AS2细胞的Topo Ⅱα、TopoⅡβ基因表达及其功能的影响.方法 设计并合成针对Topo Ⅱα和TopoⅡβ基因序列的shRNA各3对,在脂质体的介导下转染K562/AS2细胞;用荧光实时定量聚合酶链反应(PCR)分析Topo Ⅱα、TopoⅡβmRNA的表达水平;流式细胞术检测Topo Ⅱα、TopoⅡβ蛋白表达.结果 针对Topo Ⅱα-shRNA、TopoⅡβ的shRNA作用于K562/AS2细胞24 h后,Topo ⅡαmRNA水平和蛋白水平最大下调为(78.22±0.01)%、(31.17±1.27)%(P<0.05),TopoⅡβmRNA水平和蛋白水平最大下调为(57.36 ±0.01)%、(23.98 ± 1.22)%(P<0.05).结论 转染24 h后针对TopoⅡ的shRNA可抑制对ATO耐药的白血病细胞株K562/AS2细胞TopoⅡ基因的表达.     

水稻硫转运蛋白基因OsST的亚细胞定位

[目的]构建水稻硫酸根转运基因OsST与YFP黄色荧光蛋白融合表达载体,对OsST蛋白进行亚细胞定位.[方法]从水稻叶片的cDNA中克隆OsST基因ORF全长,测序验证后连入pA7-YFP表达载体,通过基因枪将融合载体转入洋葱上表皮细胞,在激光共聚焦显微镜下观察细胞中荧光发光部位.[结果]OSST蛋白定位于细胞膜和核膜上.[结论]为进一步研究硫转运蛋白的功能及硫酸根运输的机理奠定了基础.     

氯化铈对K562肿瘤细胞凋亡影响的实验研究

为了探讨氯化铈对肿瘤细胞凋亡的影响,本实验以K562细胞为研究对象,采用荧光显微镜定性检测细胞凋亡、流式细胞术定量测定细胞凋亡率.实验得出,与对照组相比,3mg/L氯化铈作用24h和500 mg/L氯化铈作用48h可以显著诱导细胞凋亡,30mg/L,150mg/L和250mg/L作用24h细胞凋亡率明显减少,同时氯化铈对K562细胞凋亡的影响随着作用时间的延长作用逐渐消失,对于同一时间来讲,氯化铈剂量和细胞凋亡之间的剂量-效应关系不明显.     

甲基化寡核苷酸灭活死亡相关蛋白激酶1基因对白血病K562细胞增殖的影响

目的 探讨互补甲基化寡核苷酸诱导灭活K562白血病细胞死亡相关蛋白激酶1基因(DAPK1)及对其增殖的影响.方法 应用Lipo2000将与DAPK1基因启动子序列互补的甲基化寡核苷酸转染进K562白血病细胞,分别应用甲基化特异性聚合酶链反应(MSP)和反转录PCR(RT-PCR)检测转染前后DAPK1基因启动子甲基化状态和mRNA表达改变.应用噻唑蓝(MTT)法检测转染前后细胞增殖变化.结果 正常组K562细胞的DAPK1基因启动子表现为未甲基化状态,可检测到相应mRNA表达;对照组寡核苷酸转染后,DAPK1基因启动子表现为未甲基化状态,mRNA表达和细胞增殖速度与正常组无明显差异;互补甲基化寡核苷酸转染后,DAPK1基因启动子呈甲基化状态,mRNA呈低表达状态,细胞增殖速度较正常组、甲基化对照寡核苷酸转染组显著增加.结论 互补甲基化寡核苷酸可诱导灭活K562白血病细胞DAPK1基因并抑制其mRNA表达,促进细胞增殖.     

增强子结合蛋白C/EBPα对白血病BALB/c裸鼠的肿瘤抑制作用

目的 探讨增强子结合蛋白C/EBPα在白血病裸鼠体内的肿瘤抑制作用.方法 将30只BALB/c裸鼠随机分转染组(10只)、空载组(10只)、对照组(10只),建立皮下瘤模型,并将30只BALB/c裸鼠如上分组建立白血病模型,将C/EBPα稳定表达细胞株pEGFP-C/EBPα-K562、空载细胞株pEGFP-K562及白血病细胞株K562分别经皮下和尾静脉注射到相应组裸鼠体内,形成皮下瘤和血液病模型.观测皮下肿瘤的变化,应用TUNEL检测细胞的凋亡,瑞特-吉姆萨染色观察血液病模型裸鼠外周血和骨髓中白血病细胞增殖能力,RT-PCR检测增殖相关基因的表达.结果 皮下瘤模型中pEGFP-C/EBPα-K562组肿瘤质量及最大直径为(24±0.1)g和(11±2)mm,空载组和对照组分别为(5.1±0.3)g、(19±3)mm和(5.7±0.4)g、(23±3)mm(均P＜0.05),TUNEL检测发现pEGFP-C/EBPα-K562组肿瘤细胞凋亡明显增加(P＜0.05);血液病模型鼠外周血可见白血病细胞,pEGFP-C/EBPα-K562组白血病细胞增殖能力明显低于空载组和对照组,可见明显细胞分化现象,RT-PCR检测发现p53基因上调和c-myc基因下调.结论 增强子结合蛋白C/EBPα在白血病小鼠体内具有促进肿瘤细胞凋亡和抑制白血病细胞增殖的能力,并能促进白血病细胞分化.C/EBPα的白血病抑制作用可能是通过对相应基因的调控来实现.     

低剂量地西他滨联合伊马替尼对K562细胞的增殖抑制作用

目的 研究低剂量地西他滨(DAC)联合伊马替尼(IM)对K562细胞株的增殖抑制作用及对bcr-abl表达的影响.方法 单药及两药联合后,通过四甲基偶氮唑蓝(MTT)法观察药物对K562细胞株的增殖抑制作用,流式细胞术检测药物对K562细胞株早期凋亡率及细胞周期,巢式反转录-聚合酶链反应(RT-PCR)半定量检测药物对K562细胞株bcr-abl mRNA表达.结果 DAC与IM单药对K562细胞的抑制作用呈浓度时间依赖性.两药联合用药抑制作用较单药组明显(F=43.947、165.580、321.193、296.101,均P<0.05),24、48、72 h各浓度组与对照组比较差异均有统计学意义(F=202.759、168.457、417.538,均P<0.05).DAC及IM单药作用药物对K562细胞株均使G,期细胞明显增多,IM0.2μmol/L作用于K562细胞株48 h可见6.7%早期凋亡细胞,IM 0.2 μmol/L联合DAC 4μmol/L早期凋亡细胞增加至8.4%.bcr-abl mRNA表达水平降低,DAC 4 μmol/L作用48 h后可降低K562细胞中bcr-abl mRNA表达(约14%),IM 0.2 μmol/L降低约40%,联合用药表达量明显降低(约60%).联合用药组与单药组比较差异有统计学意义(F=71.981,P<0.05).结论 DAC对K562细胞的增殖抑制作用与细胞周期阻滞、诱导凋亡及降低bcr-abl mRNA表达有关,两药联合可显著抑制K562细胞增殖.     

雷公藤红素体外抑制人类急性髓系白血病细胞的实验研究

目的 研究雷公藤红素体外对人类急性髓系白血病(AML)细胞的体外抑制作用.方法 应用四甲基偶氮唑蓝(MTT)法和流式细胞术(FCM)等方法研究雷公藤红素对人类AML细胞的影响.结果 MTT实验显示,与空白组比较,培养不同时间后不同浓度组雷公藤红素的细胞增殖抑制率均明显升高,差异有统计学意义(P＜0.01).FCM检测结果显示,1 μmol/L以上浓度雷公藤红素作用不同时间,AML细胞均可出现凋亡现象,凋亡率明显高于不加药物的对照组(P＜0.01).结论 雷公藤红素对AML有明显的抑制作用,其作用可能与其诱导细胞凋亡有关.     

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells

The DNA sequences of the recA gene from 25 strains of bacteria are known. The evolution of these recA gene sequences, and of the derived RecA protein sequences, is examined, with special reference to the effect of variations in genomic G + C content. From the aligned RecA protein sequences, phylogenetic trees have been drawn using both distance matrix and maximum parsimony methods. There is a broad concordance between these trees and those derived from other data (largely 16S ribosomal RNA sequences). There is a fair degree of certainty in the relationships among the "Purple" or Proteobacteria, but the branching pattern between higher taxa within the eubacteria cannot be reliably resolved with these data.     

Contrasting patterns of regulation of the antioxidant selenoproteins, thioredoxin reductase, and glutathione peroxidase, in cancer cells

There is strong evidence that selenium protects against certain human cancers, but the underlying mechanism is unknown. Glutathione peroxidase (GPX1) and thioredoxin reductase (TR), the most abundant antioxidant selenium-containing proteins in mammals, have been implicated in this protection. We analyzed the expression of TR and GPX1 in the following model cancer systems: (1) liver tumors in TGFalpha/c-myc transgenic mice; (2) human prostate cell lines from normal and cancer tissues; and (3) p53-induced apoptosis in a human colon cancer cell line. TR was induced while GPX1 was repressed in malignancies relative to controls in transgenic mice and prostate cell lines. In the colon cell line, p53 expression resulted in elevated GPX1, but repressed TR. The data indicate that TR and GPX1 are regulated in a contrasting manner in the cancer systems tested and reveal the p53-dependent regulation of selenoprotein expression. The data suggest that additional studies on selenoprotein regulation in different cancers are required to evaluate future implementation of selenium as a dietary supplement in individuals at risk for developing certain cancers.     

白藜芦醇诱导白血病Molt-4细胞凋亡及对WAVE1基因表达的影响

目的 探讨白藜芦醇诱导人类急性淋巴细胞白血病Molt-4细胞凋亡的作用机制.方法 噻唑蓝比色法(MTF)测定细胞生长抑制率;流式细胞术检测细胞周期分布及凋亡率;半定量反转录聚合酶链反应(RT-PCR)法检测WAVE1基因的表达.结果 MTT结果显示:12.5、25.0、50.0、100.0、200.0 μmol/L的白藜芦醇作用于Moh-4细胞24、48、72 h后,细胞的最大抑制率分别达到29.32%、36.11%、53.92%、62.50%、74.98%,并呈时间-剂量依赖性(F=33.614,P<0.05);流式细胞术检测结果显示:与对照组相比,50.0、100.0 μmol/L白藜芦醇作用于Molt-4细胞48 h后,S期细胞比例由42.2%分别上升为68.6%和78.1%,细胞发生了S期阻滞(F=19.453,P<0.01);PCR结果显示:50.0、100.0 μmol/L的白藜芦醇处理Molt-4细胞48 h后,WAVE1/GAPDH比值分别为0.356±0.03、0.382±0.05,与对照组(0.586±0.06)比较,差异有统计学意义(F=8.950,P<0.01).结论 白藜芦醇可诱导Moh-4细胞发生凋亡,其作用机制可能与下调WAVE1基因表达有关.     

Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.     

Trimidox-mediated morphological changes during erythroid differentiation is associated with the stimulation of hemoglobin and F-cell production in human K562 cells

Previous studies have demonstrated that stimulation of the ventral hippocampal (VH) formation (including the ventral CA1 and subicular areas) elicits increased locomotor activity in rats. The locomotor-activating effects of VH stimulation have been hypothesized to be mediated via hippocampal output to cortical and subcortical dopamine (DA) systems. This study examined whether increased locomotor activity produced by VH stimulation was blocked by pretreatment with a DA receptor antagonist, and whether DA metabolism in subdivisions of the nucleus accumbens, caudate-putamen, and prefrontal cortex was elevated by VH stimulation. Stimulation of the VH (defined as the ventral CA1 and its borders, ventral subiculum, and entorhinal cortex) with the cholinergic agonist carbachol was found to elevate locomotor activity, while pretreatment with the D2 receptor antagonist haloperidol blocked this effect. Stimulation of the VH did not alter DA metabolism (i.e., ratio of the DA metabolites DOPAC or HVA/DA) in any of the brain regions studied. These results indicate that the increased locomotor activity elicited by VH stimulation is not associated with dramatic increases in DA metabolism, but that it does require tonic activation of D2 receptors.