NME3蛋白在急性髓系白血病细胞株HL-60和患者骨髓表达趋势研究

为了验证HL-60细胞向粒、单两系分化前后非转移细胞蛋白3（NME3）的表达差异,并探讨该蛋白对急性髓系白血病的诊断价值,采用全反式维甲酸（ATRA）和甾体类新药NSC67657分别诱导急性髓系白血病HL-60细胞向粒系和单核系分化,通过细胞化学染色判断细胞分化方向,通过细胞表面分化抗原（CD11b／CD14）检测判断细胞分化程度,通过磷脂酰丝氨酸外翻排除细胞凋亡,建立细胞分化模型,应用RT-PCR和Western blot方法验证NME3基因和蛋白在细胞分化前后的差异表达情况。收集26例临床确诊的各种类型髓系白血病患者及5例正常人骨髓样本,比较分析NME3蛋白在不同样本组中的表达趋势。结果表明：ATRA（2μmol／L,5d）和NSC67657（10μmol／L,5d）可分别诱导HL-60细胞向粒系和单核系分化,其分化程度达到90％以上且不伴随凋亡发生,NME3基因与蛋白表达在细胞两系分化后明显下降。对患者骨髓有核细胞分析发现,NME3蛋白在急性髓系白血病患者骨髓样本中表达较慢性髓系白血病患者组及正常对照组明显升高。结论：NME3蛋白在HL-60细胞分化后表达下调．与其在患者样本中的袁达趋蛰相符．提示该蛋白可能作为鱼性醋系白血痛潜在的分平标点物。     

鼠尾草酸对NB4细胞生长及凋亡、分化作用的体外研究

目的 观察鼠尾草酸（Carnosic acid,CA）对NB4细胞生长及凋亡、分化的诱导作用.方法 通过四氮唑蓝比色（MTT）,细胞形态,流式细胞仪测定细胞周期、凋亡率及CD14的表达,观察鼠尾草酸对NB4细胞的影响.结果 NB4细胞经2.5μmol/L以上浓度的鼠尾草酸作用后增殖受到抑制,2.5μmol/L、5μmol/L、10μmol/L CA 作用72h后,细胞形态呈现凋亡细胞的特征.2.5μmol/L、5μmol/L、10μmol/L CA 作用于NB4细胞72h凋亡率分别为7.216%、11.131 %、20.336%,与对照组相比差异显著,G0/G1期细胞阻滞.CD14的表达与对照组相比无差异性.结论 CA对NB4细胞有生长抑制作用,能诱导NB4细胞发生凋亡,具有一定的量效和时效关系,单独使用CA的分化作用不显著.     

Recombinant human CD19L-sTRAIL effectively targets B cell precursor acute lymphoblastic leukemia

Patients with B cell precursor acute lymphoblastic leukemia (BPL) respond well to chemotherapy at initial diagnosis; however, therapeutic options are limited for individuals with BPL who relapse. Almost all BPL cells express CD19, and we recently cloned the gene encoding a natural ligand of the human CD19 receptor (CD19L). We hypothesized that fusion of CD19L to the soluble extracellular domain of proapoptotic TNF-related apoptosis-inducing ligand (sTRAIL) would markedly enhance the potency of sTRAIL and specifically induce BPL cell apoptosis due to membrane anchoring of sTRAIL and simultaneous activation of the CD19 and TRAIL receptor (TRAIL-R) apoptosis signaling pathways. Here, we demonstrate that recombinant human CD19L-sTRAIL was substantially more potent than sTRAIL and induced apoptosis in primary leukemia cells taken directly from BPL patients. CD19L-sTRAIL effectively targeted and eliminated in vivo clonogenic BPL xenograft cells, even at femtomolar-picomolar concentrations. In mice, CD19L-sTRAIL exhibited a more favorable pharmacokinetic (PK) profile than sTRAIL and was nontoxic at doses ranging from 32 fmol/kg to 3.2 pmol/kg. CD19L-sTRAIL showed potent in vivo antileukemic activity in NOD/SCID mouse xenograft models of relapsed and chemotherapy-resistant BPL at nontoxic fmol/kg dose levels. Together, these results suggest that recombinant human CD19L-sTRAIL has clinical potential as a biotherapeutic agent against BPL.     

辛伐他汀对急性单核细胞白血病SHI-1细胞增殖和凋亡的影响

本研究探讨辛伐他汀(simvastatin,SIM)对急性单核细胞白血病SHI-1细胞增殖和凋亡的影响及其作用机制。用MTT法检测不同浓度SIM作用不同时间对SHI-1细胞生长的抑制作用;实验分为阴性对照及辛伐他汀处理组(终浓度分别为5、10、20μmol/L),处理48小时后,用流式细胞仪检测细胞凋亡及周期分布;半定量RT-PCR法检测caspase-3、BCL-2mRNA的表达;Western blot法检测caspase-3、BCL-2蛋白表达。结果表明,辛伐他汀对SHI-1细胞的生长有明显抑制作用,且呈时间与剂量依赖性。与对照组相比,辛伐他汀处理SHI-1细胞48小时后,各浓度组S期细胞百分比明显增多,5μmol/L辛伐他汀处理SHI-1细胞能明显阻滞细胞周期进程,但不能诱导细胞凋亡。细胞中BCL-2mRNA表达随辛伐他汀浓度增加而下降,caspase-3mRNA表达随辛伐他汀浓度增加而增高(p<0.05)。SHI-1细胞中BCL-2蛋白表达随辛伐他汀浓度升高而降低,caspase-3蛋白表达随辛伐他汀浓度升高而增加(p<0.05)。结论 :辛伐他汀能抑制SHI-1细胞增殖并诱导其凋亡,其机制可能与其下调BCL-2和上调caspase-3表达有关。     

Effect of Embelin on proliferation, differentiation and aopotosis of HL-60 cells

OBJECTIVE: To study the effect of embelin on proliferation, differentiation and apoptosis of HL-60 cells and explore its possible mechanism. METHODS: Different concentration of embelin were used to treat HL-60 cells. Cell growth curve was analysed by MTT assay, cell apoptosis by Annexin V/PI double staining and JC-1 dye. The differentiation of HL-60 cells was evaluated by expression of CD33, CD34, CD11b and CD14. Bone marrow cells (BMC) from nine patients with acute nonlymphocytic leukemia (ANML) were also studied. RESULTS: Embelin induced differentiation of HL-60 cells with significant increase of CD14 and CD11b expression at 33.97μmol/L for 3 days (P < 0.01). Embelin induced apoptosis of HL-60 cells in a time- and dose-dependent manner, the apoptosis rates were (9.23 ± 0.05)%, (25.86 ± 0.30)% and (39.03 ± 0.07)% respectively at 339.67 μmol/L of embelin for 12-, 24- and 48-hours treatment (P < 0.05); the apoptosis rates were (0.07 ± 0.03)%, (7.43 ± 0.30)%, (14.01 ± 0.01)%, (25.52 ± 0.03)% and (39.15 ± 0.01)% respectively at 10.19, 33.97, 101.90, 339.67 and 1019.02 μmol/L of embelin for 24-hours culture (P < 0.05). Clusters of differentiation antigen on BMC from three acute promyelocytic leukemia patients showed significant changes at 33.97 μmol/L of embelin treatment for 3 days. Embelin induced apoptosis of BMCs from all the nine ANML patients at 33.97 μmol/L for 24 hour. CONCLUSION: Embelin can inhibit proliferation and induce differentiation and apoptosis of HL-60 cells. The mechanism may be related to mitochondrial apoptosis pathway. Embelin at subtoxic concentration doesn't promote leukemia BMC differentiation, but at 339.67 μmol/L induces apoptosis of these cells.     

Embelin对HL-60细胞增殖、分化和凋亡的影响

目的 研究Embelin对HL-60细胞增殖、分化和凋亡的影响,并对其作用机制进行初步探讨.方法 用不同浓度的Embelin处理HL-60细胞,采用MTT法绘制生长曲线,通过Annexin V/PI 复染及JC-1染色,观察Embelin对HL-60细胞凋亡的影响;通过流式细胞术检测细胞表面分化抗原CD33、CD34、CD11b和CD14表达的变化.在对HL-60细胞研究的基础上,选择9例急性非淋巴细胞白血病(ANLL)患者的骨髓进行相应的研究.结果 Embelin对HL-60细胞具有增殖抑制作用,且作用呈时间和剂量依赖性.24 h的,IC50值为429.98 μmoL/L.33.97 μmol/L的Embelin作用HL-60细胞3 d,流式细胞术检测结果 显示CD11b、CD14阳性细胞率均升高(P值均<0.01);339.67 μmol/L Embelin作用于HL-60细胞后12、24及48 h的特异性凋亡率分别为(9.23 ±0.05)%、(25.86 ±0.30)%和(39.03±0.07)%,10.19、33.97、101.90、339.67及1019.02 μmol/L Embelin作用于HL-60细胞24 h后特异性凋亡率分别为(0.07±0.03)%、(7.43±0.30)%、(14.01±0.01)%、(25.52±0.03)%和(39.15±0.01)%,其诱导凋亡的作用呈时间及剂量依赖性.33.97 μmol/L的Embelin作用于ANLL 患者骨髓细胞3 d,3例M3患者骨髓细胞表面分化抗原出现显著性改变;339.67 μmol/L的Embelin作用患者骨髓细胞24 h,9例均发生凋亡.结论 亚细胞毒浓度的Embelin可诱导HL-60细胞向单核细胞分化;高浓度的Embelin对HL-60细胞具有增殖抑制及促进凋亡的作用.其促进凋亡的机制与线粒体凋亡途径有关.亚细胞毒浓度的Embelin对体外培养的M3患者骨髓细胞具有促进分化的作用;339.67 μmol/L的Embelin对ANLL骨髓细胞具有促进凋亡的作用.     

MG132对初治、复发及耐药急性白血病细胞体外诱导凋亡作用及NF-κB活性改变对survivin基因表达的影响

目的 观察MG132对初治、复发及耐药急性白血病细胞体外诱导凋亡作用及NF-κB活性改变对survivin基因表达的影响.方法 分别采集32例(初治12例、复发10例及耐药10例)急性白血病患者骨髓,经Ficoll分离出单个核细胞体外培养,加入不同浓度的MG132培养24 h,Annexin V-FITC检测细胞凋亡,Western blotting和RT-PCR法检测NF-κB p65和survivin的表达.结果 不同浓度的MG132可诱导初治、复发及耐药急性白血病细胞发生凋亡,且呈剂量依赖效应.NF-κB活性和survivin表达在初治组和复发组、初治组和耐药组之间有显著性差异(P<0.01),但在复发组和耐药组间无显著性差异(P>0.05).survivin表达与NF-κB活性密切正相关(Pearson相关系数为0.957,P<0.01),survivin表达随着NF-κB活性被抑制而下调.结论 MG132在体外可以通过选择性抑制NF-κB活性诱导初治、复发及耐药急性白血病细胞凋亡,并下调抗凋亡基因survivin的表达.     

甘草酸单铵盐调节急性早幼粒细胞白血病细胞NB4肿瘤干细胞样特性、氧化应激及线粒体的功能

目的:探讨甘草酸单铵盐(MAG)对急性早幼粒细胞白血病细胞NB4肿瘤干细胞样特性、氧化应激及线粒体功能的影响.方法:CCK-8法检测急性早幼粒细胞白血病细胞NB4活力,筛选合适剂量;克隆法检测NB4细胞增殖;Western blot检测细胞周期相关蛋白表达;流式细胞术检测细胞凋亡并分选NB4干细胞阳性(CD133+);...     

地西他滨对全反式维甲酸耐药细胞株NB4-R2的增殖抑制及诱导凋亡作用研究

本研究探讨地西他滨(DAC)对全反式维甲酸耐药细胞株NB4-R2的增殖抑制及诱导凋亡作用。应用细胞增殖及毒性检测法(新型四唑单钠盐法)观察0.01-0.5μmol/L DAC作用于NB4-R2细胞24、48及72 h后的细胞增殖活力变化;PI单染及AnnexinⅤ-FITC/PI双染流式细胞术观察不同浓度DAC(0.05-5μmol/L)对NB4-R2处理48 h后的细胞凋亡率;RT-PCR法检测编码P糖蛋白(P-gp)的多药耐药基因1(MDR1)转录水平的变化。结果表明,0.01-0.5μmol/L DAC可抑制NB4-R2细胞增殖,并呈现明显的量-效与时-效关系;作用48及72 h后的IC50分别为0.089和0.064μmol/L;0.05-5μmol/L DAC以浓度依赖的方式诱导NB4-R2细胞凋亡,下调MDR1基因表达。结论:低浓度DAC(<0.5μmol/L)对NB4-R2细胞有增殖抑制作用,较高浓度DAC(1、5μmol/L)可以诱导NB4-R2细胞凋亡,同时使MDR1表达下调。     

丝氨酸/苏氨酸蛋白激酶抑制剂治疗成人急性白血病的研究

目的应用基因表达谱芯片来检测丝氨酸/苏氨酸蛋白激酶Raf-1抑制剂对急性髓系白血病（AML）细胞基因表达的影响,试图进一步探讨丝氨酸/苏氨酸蛋白激酶Raf-1抑制剂抗白血病细胞增殖的机制。方法利用cDNA基因芯片研究10例成人AML,用两种不同的荧光素cy3和cy5通过逆转录反应将白血病细胞经丝氨酸/苏氨酸蛋白激酶Raf-1抑制剂作用前后的RNA分别标记成两种探针,并与基因表达谱芯片进行杂交,通过计算机扫描分析得出这些基因在经丝氨酸/苏氨酸蛋白激酶Raf-1抑制剂处理前后的表达差异。结果基因表达谱分析两次重复实验结果,最后筛选出包括与细胞凋亡、细胞周期等相关表达差异的基因共20条,其中12条表达上调,8条表达下调。结论结果提示丝氨酸/苏氨酸蛋白激酶Raf-1抑制剂抗成人急性粒细胞白血病的作用机制与抑制白血病细胞周期和诱导白血病细胞凋亡有很大关系。     

醋酸棉酚诱导淋巴细胞白血病细胞凋亡的作用及联合地塞米松的效应

本研究旨在探讨醋酸棉酚诱导人急、慢性淋巴细胞白血病细胞凋亡的作用及联合地塞米松的效应。用流式细胞仪检测醋酸棉酚对人急、慢性淋巴细胞白血病原代细胞凋亡的影响。用MTT比色法测定醋酸棉酚对人Burkitt淋巴瘤细胞株Raji细胞及正常骨髓单个核细胞存活率的影响。用流式细胞仪检测醋酸棉酚与地塞米松联合诱导Raji细胞的凋亡率。结果表明,≥5μmol/L醋酸棉酚能诱导原代培养的人急性淋巴细胞白血病细胞凋亡,呈浓度及时间依赖性。醋酸棉酚对原代培养的慢性淋巴细胞白血病细胞诱导凋亡的浓度更高,50μmol/L醋酸棉酚作用48 h,急、慢性淋巴细胞白血病细胞的凋亡率分别为(90.4±6.2)%和(51.7±10.3)%。≤10μmol/L醋酸棉酚对正常骨髓单个核细胞的存活率没有明显影响,醋酸棉酚作用48和72 h,对正常骨髓单个核细胞的IC50值分别为Raji细胞的7.1和9.1倍。≥10μmol/L地塞米松能诱导Raji细胞凋亡,不同浓度地塞米松与10μmol/L醋酸棉酚联用,Raji细胞凋亡率明显增加(P<0.05)。结论:醋酸棉酚能诱导原代培养的人急、慢性淋巴细胞白血病细胞凋亡;对正常骨髓单个核细胞的抑制作用明显低于Raji细胞;与地塞米松联用可增强后者诱导Raji细胞凋亡的效应。     

AMN107联合血红素加氧酶-1抑制剂诱导慢性髓系白血病细胞凋亡

运用AMN107(尼洛替尼)联合血红素加氧酶-1(HO-1)抑制剂锌原卟啉Ⅸ(ZnPPⅨ)作用于慢性髓系白血病(CML)细胞株K562细胞,研究其对CML细胞增殖的影响并探讨其作用机制。采用MTT法和台盼蓝染色法检测AMN107(10μmol/L)及ZnPPⅨ(10μmol/L)单独或联合处理不同时间的细胞增殖率;半定量RT-PCR法和Westernblot法检测空白对照组、ZnPPⅨ(10μmol/L)组、AMN107(10μmol/L)组、AMN107(10μmol/L)联合ZnPPⅨ(10μmol/L)组48 h时细胞HO-1的表达;AnnexinⅤ/PI双染色法检测处理48 h时各组细胞凋亡情况。结果表明:联合用药对细胞的抑制作用最强,且呈时间依赖性;联合用药组HO-1的表达量最低;空白对照组、ZnPPⅨ(10μmol/L)组、AMN107(10μmol/L)组、AMN107(10μmol/L)联合ZnPPⅨ(10μmol/L)组48 h时细胞凋亡率分别为(11.38±0.02)%、(17.44±0.08)%、(39.81±0.07)%和(56.46±0.19)%。结论:第二代酪氨酸激酶抑制剂AMN107具有诱导CML细胞凋亡的作用;抑制HO-1表达能加强AMN107对CML细胞的杀伤作用,这为临床进一步提高CML的疗效提供实验依据。     

5-氮杂胞苷对骨髓瘤细胞增殖抑制作用及对XAF1基因表达的影响

目的 探讨5-氮杂胞苷处理对骨髓瘤细胞系RPMI 8226和XG-7细胞中XAF1基因表达的影响及体外抗骨髓瘤作用的机制.方法 采用逆转录PCR和Western blot方法检测骨髓瘤细胞系RPMI 8226和XG-7细胞中XAF1基因和蛋白的表达.采用甲基化特异性PCR(MSP)方法检测XAFI基因启动子CpG岛甲基化状态.采用0～5 μmoL/L 5-氮杂胞苷处理骨髓瘤细胞株.采用CCK-8比色法检测5-氮杂胞苷处理对骨髓瘤细胞增殖抑制作用.采用Annexin V/7-AAD染色流式细胞术检测细胞凋亡.结果 XG-7细胞不表达XAF1 mRNA及蛋白,RPMI 8226细胞表达XAF1 mRNA转录本1和2.XG-7和RPMI 8226细胞XAFl基因启动子CpG岛均存在过甲基化.XG-7和RPMI 8226细胞经2.5μmol/L 5-氮杂胞苷处理72 h后仅表达XAF1 mRNA转录本1并表达XAF1蛋白,并且XAF1基因启动子CpG岛甲基化程度降低.5-氮杂胞苷抗骨髓瘤作用呈时间和浓度依赖性.5-氮杂胞苷处理RPMI 8226和XG-7细胞48 h的IC50值分别为2.4 μmol/L和2.6 μmol/L.结论 骨髓瘤细胞中抑癌基因XAFI表达缺失或表达异常与XAF1基凶启动子CpG岛过甲基化有关.5-氮杂胞苷处理可以诱导XAF1 mRNA及蛋白表达.5-氮杂胞苷在临床上能达到的药物浓度下具有抗骨髓瘤作用,其作用机制是诱导骨髓瘤细胞凋亡.     

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。     

抑制NAMPT表达增强K562细胞对伊马替尼的敏感性及相关机制研究

本研究探讨烟酰胺磷酸核糖转移酶(NAMPT)对K562细胞(人慢性髓系白血病细胞株)对伊马替尼敏感性的影响及其相关机制。在体外合成针对NAMPT基因的小干扰RNA(siRNA),转染K562细胞株。将转染后的K562细胞经1μmol/L伊马替尼作用48 h后,采用PI/Calcein染色方法测定细胞存活率;经不同浓度伊马替尼作用48 h后,采用MTS法测定细胞增殖活性,计算半数抑制浓度(IC50)。未经转染的K562细胞经1μmol/L伊马替尼作用3-48 h后,采用Western blot方法检测NAMPT表达的变化。通过对NCBI GEO中基因表达数据的挖掘分析及Western blot方法检测,探讨NAMPT-siRNA和伊马替尼对凋亡相关基因表达的影响。采用荧光素酶报告基因技术,研究NAMPT-siRNA、伊马替尼对NF-κB转录活性的影响。结果表明,1μmol/L伊马替尼作用于K562细胞48 h对NAMPT表达无明显影响。NAMPT-siRNA能够明显抑制K562细胞NAMPT的表达,再经1μmol/L伊马替尼作用后,NAMPT-siRNA干扰组细胞存活率明显降低(P<0.05),提示抑制NAMPT表达能增强K562细胞对伊马替尼的敏感性。不同浓度伊马替尼作用后,干扰组IC50值明显降低(P<0.05);拟合增殖曲线在0.01-0.1μmol/L伊马替尼浓度范围内斜率增大,提示在此范围内NAMPT-siRNA与伊马替尼的协同作用最强。基因表达谱分析及Western blot验证结果都显示,NAMPT-siRNA和伊马替尼处理均可下调NF-κB下游因子抗凋亡基因Bcl-2表达水平,两者同时作用时效果更明显。NAMPT-siRNA及伊马替尼均能降低NF-κB转录活性,两者同时作用效果更显著。结论:伊马替尼可能并非通过调节NAMPT表达影响细胞存活;抑制NAMPT表达能增强K562细胞对伊马替尼的敏感性,可能与抑制NF-κB及其下游因子Bcl-2有关。     

Apogossypolone诱导骨髓瘤细胞凋亡及其机制研究

本研究探讨棉酚衍生物apogossypolone（ApoG2）对多发性骨髓瘤细胞系的抑制增殖、诱导凋亡的作用及其作用机制。应用台盼蓝染色、Hoechst 33258染色、透射电子显微镜检、DNA凝胶电泳法、流式细胞仪分析细胞DNA含量等来判断ApoG2对多发性骨髓瘤细胞的抑制增殖和诱导凋亡作用。以caspase-3、caspase-9以及BCL-2、BCL—XL分析ApoG2诱导骨髓瘤细胞凋亡的可能机制。结果表明：ApoG2对多发性骨髓瘤细胞增殖具有明显的抑制作用,IC50值在U266细胞和Wusl细胞（48小时）分别为0．1、0．2μmol／L。ApoG2可以诱导骨髓瘤细胞凋亡且呈时间和剂量依赖性。ApoG2可以使骨髓瘤细胞周期停止在G2期,U266细胞G2期比例从9．7％增至19．6％。Wusl细胞从9．8％增至31．7％。ApoG2能导致U266、Wusl细胞caspase-9、caspase-3活性增高。U266细胞和Wusl细胞经ApoG2 25μmoL／L作用24小时后,BCL—XL表达分别下降（7．3±1．4）％和（11．2±6．2）％,Wusl细胞BCL-2表达下降84．4±3．4％。结论：ApoG2对多发性骨髓瘤细胞具有抑制增殖和诱导凋亡作用,其作用机制可能和下调BCL-2／BCL—XL表达以及影响细胞周期有关。     

Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling

Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.     

Antitumor activity of ER-37328, a novel carbazole topoisomerase II inhibitor

DNA topoisomerase II has been shown to be an important therapeutic target in cancer chemotherapy. Here, we describe studies on the antitumor activity of a novel topoisomerase II inhibitor, ER-37328 [12,13-dihydro-5-[2-(dimethylamino)ethyl]-4H-benzo[c]pyrimido[5,6,1- jk]carbazole-4,6,10(5H,11H)-trione hydrochloride]. ER-37328 inhibited topoisomerase II activity at 10 times lower concentration than etoposide in relaxation assay and induced double-strand DNA cleavage within 1 h in murine leukemia P388 cells, in a bell-shaped manner with respect to drug concentration. The maximum amount of DNA cleavage was obtained at 2 microM. Like etoposide, ER-37328 (2 microM) induced topoisomerase II-DNA cross-linking in P388 cells. A spectroscopic study of ER-37328 mixed with DNA demonstrated that ER-37328 has apparent binding activity to DNA. ER-37328 showed potent growth-inhibitory activity against a panel of 21 human cancer cell lines [mean (50% growth-inhibitory concentration) GI50 = 59 nM]. COMPARE analysis according to the National Cancer Institute screening protocol showed that the pattern of the growth-inhibitory effect of ER-37328 was similar to that of etoposide, but different from that of doxorubicin. Studies on etoposide-, amsacrine [4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)]-, and camptothecin-resistant P388 cell lines showed that: (a) etoposide- and m-AMSA-resistant P388 cell lines were partially resistant to ER-37328 compared with the parental cell line; and (b) a camptothecin-resistant cell line showed no cross-resistance to ER-37328. In addition, ER-37328 overcame P-glycoprotein-mediated resistance. In vivo, ER-37328 produced potent tumor regression of Colon 38 carcinoma inoculated s.c., and its activity was superior to that of etoposide or doxorubicin. These results indicate that ER-37328 inhibits topoisomerase II activity through the formation of topoisomerase II-DNA cleavable complex and has potent antitumor activity both in vitro and in vivo.     

ENMD-2076 is an orally active kinase inhibitor with antiangiogenic and antiproliferative mechanisms of action

ENMD-2076 is a novel orally active, small molecule kinase inhibitor with a mechanism of action involving several pathways key to tumor growth and survival: angiogenesis, proliferation, and the cell cycle. ENMD-2076 has selective activity against the mitotic kinase Aurora A, as well as kinases involved in angiogenesis (VEGFRs, FGFRs). ENMD-2076 inhibited the growth in vitro of a wide range of human solid tumor and hematopoietic cancer cell lines with IC(50) values ranging from 0.025 to 0.7 μmol/L. ENMD-2076 was also shown to induce regression or complete inhibition of tumor growth in vivo at well-tolerated doses in tumor xenograft models derived from breast, colon, melanoma, leukemia, and multiple myeloma cell lines. Pharmacodynamic experiments in vivo showed that in addition to inhibiting Aurora A, single doses of ENMD-2076 had sustained inhibitory effects on the activation of Flt3 as well as the angiogenic tyrosine kinases, VEGFR2/KDR and FGFR1 and 2. ENMD-2076 was shown to prevent the formation of new blood vessels and regress formed vessels in vivo at doses equivalent to those that gave substantial activity in tumor xenograft models. These results indicate that ENMD-2076 is a well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic/antiproliferative profile and provides strong preclinical support for use as a therapeutic for human cancers. Several phase 1 studies involving ENMD-2076 have been recently completed, and the compound is currently being evaluated in a phase 2 clinical trial in patients with platinum-resistant ovarian cancer.     

ZnPcH1介导的光动力疗法净化白血病SHI-1细胞

本研究探讨新型酞菁类光敏剂二磺基二邻苯二甲酰亚胺甲基酞菁锌(ZnPcH1)介导的光动力疗法(PDT)对急性单核细胞白血病SHI-1细胞的杀伤作用及其机制,为PDT应用于白血病自体骨髓体外净化提供理论依据。以SHI-1细胞作为研究对象,应用MTT比色法检测细胞增殖。采用AO/EB复合染色、TUNEL、DNA二倍体分析、An-nexin-Ⅴ-FITC/PI双染法等分析细胞的死亡方式。SHI-1细胞与正常骨髓单个核细胞(MNC)以1∶100-1∶10 000混合建立混合细胞模型,通过巢式RT-PCR检测SHI-1细胞融合基因MLL/AF6 mRNA的表达以研究ZnPcH1-PDT对骨髓MNC中所掺入的SHI-1细胞的净化效果。结果表明,ZnPcH1-PDT对SHI-1细胞有杀伤作用,且呈量效关系。Zn-PcH1-PDT能诱导细胞凋亡,且呈时间依赖性。0.5μmol/L ZnPcH1-PDT可完全杀灭模拟缓解骨髓中掺入的SHI-1细胞。结论:ZnPcH1-PDT有希望成为新的高效而简便的骨髓净化手段。