Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

曲古菌素A对HL-60细胞组蛋白乙酰化水平和凋亡的作用

本研究旨在探讨曲古菌素A(trichostatinA ,TSA)高效低毒的抗癌机理。应用细胞培养、MTT法 ,免疫细胞化学技术和Annexin V FITC PI双标流式细胞术观察TSA对HL 6 0细胞和正常人外周血单个核细胞 (normalhumanperipheralbloodmononuclearcell,NPBMNC)的生长抑制 ,组蛋白乙酰化水平以及诱导凋亡的影响。结果表明 :TSA能够以时间、剂量依赖性方式抑制HL 6 0细胞增殖 ,36小时IC50 为 10 0ng ml。TSA能够诱导HL 6 0细胞凋亡 ,其作用也呈时间、剂量依赖性。TSA在显著诱导HL 6 0细胞凋亡的浓度和时间范围内对NPBMNC无明显的诱导凋亡作用。TSA处理 4小时后 ,HL 6 0细胞和NPBMNC组蛋白乙酰化水平上调显著高于未用TSA各组 (P<0 .0 5 ) ,但两者之间无显著性差异 (P >0 .0 5 )。结论 :TSA对HL 6 0细胞有明确的抑制增殖能力 ;TSA有明确的诱导HL 6 0细胞凋亡的能力 ,这可能是TSA体外抑制白血病细胞系HL 6 0生长和发挥抗白血病作用的主要机理之一 ;与NPBMNC相比较 ,TSA能够选择性诱导HL 6 0细胞凋亡 ;TSA选择性抑制HL 6 0细胞的机理与TSA调控HL 6 0细胞和NPBMNC组蛋白乙酰化水平的差异无关。     

1例难治性原发中枢弥漫大B细胞淋巴瘤CAR-T细胞治疗中的多学科联合诊治

本文介绍1例原发中枢弥漫大B细胞淋巴瘤合并重症感染并气管插管患者的多学科联合诊治(MDT)过程。该患者初期起病隐匿,诊断困难,辗转于神经内科、神经外科、血液科就诊,经过多次腰椎穿刺检查后最终确诊原发中枢神经系统弥漫大B细胞淋巴瘤,给予多次化疗后未能获得完全缓解,且合并重症感染并行气管插管治疗,患者再次转至我院血液科治疗。经过我院MDT讨论,为患者制定了一个全面详尽的治疗方案,包括桥接化疗、CAR-T细胞治疗,在此过程中淋巴瘤获得完全缓解,患者一般状况明显好转。该患者的诊治过程体现了MDT在难治性原发中枢神经系统弥漫大B细胞淋巴瘤患者中,尤其是合并复杂并发症的患者中存在重要的价值,对于明确患者诊断、提供最佳治疗方案、改变预后结局,发挥着决定性的作用。     

Effects of Triptolide on Cell Proliferation and CXCR4 Expression in Burkitt＇s Lymphoma Raji Cells In Vitro

Objective： To investigate the inhibitory effects of triptolide on cell proliferation and CXCR4 expression in Burkitt＇s lymphoma cell line Raji cells. Methods： The effects of triptolide on the growth of Raji cells were studied by 3-（4, 5-Dimethyl-2-thiazolyl）-2, 5-diphenyl-2H-tetrazolium（MTT） assay. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α （rhSDF-1α） in vitro. Results： Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. Triptolide could downregulate the CXCR4 expression on Raji cells in a dose-dependent manner. Furthermore, chemotaxis assays showed that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent. Conclusion： Triptolide could inhibit the proliferation and migration of Raji cells in vitro. The underlying anti-tumor mechanism of triptolide might be related to the anti-proliferative effect and the blockage of SDF-1/CXCR4 axis.     

藤黄酸对急性白血病细胞U937增殖和凋亡的影响及对核孔蛋白Nup88的调控作用

目的 观察藤黄酸对白血病细胞株U937增殖和凋亡的影响及其对核孔蛋白Nup88的调控作用,研究藤黄酸诱导凋亡作用与其调节Nup88的相互关系.方法 采用MTT比色法检测细胞增殖活性,Annexin-V FITC/PI双标法及Hoechst 33258染色法分析细胞凋亡的改变,流式细胞术分析细胞周期和细胞内核孔蛋白Nup88的改变,RT-PCR检测藤黄酸对白血病细胞内Nupp88基因表达的调控作用;共聚焦显微镜观察Nup88蛋白的细胞分布情况.结果 藤黄酸能明显抑制U937细胞的增殖,其抑制作用呈时间、剂量依赖性,其24 h的IC50为(1.019±0.134)mg/L;此外,藤黄酸还具有诱导U937细胞凋亡和G0/G1期阻滞的作用.核孔蛋白Nup88弥漫分布于白血病细胞的核浆之间,以细胞浆和核膜为主,经藤黄酸干预后,Nup88的蛋白和mRNA表达水平明显下降,主要集中于核膜的胞浆面,偶见胞浆中表达.结论 藤黄酸能明显抑制U937白血病细胞的增殖,并诱导其凋亡.而藤黄酸诱导的核孔蛋白Nup88的重新分布以及表达量的下调可能参与了其诱导凋亡作用.     

肿瘤坏死因子α对Raji细胞Notch1蛋白表达的影响

目的 通过探讨肿瘤坏死因子α(TNF-α)作用淋巴瘤细胞系Raji细胞后Notch1蛋白表达的变化,进一步明确TNF-α对淋巴瘤细胞Notch信号途径的影响.方法 采用半定量RT-PCR方法检测Raji细胞Notch1 mRNA的含量;用流式细胞术分析Raji细胞Notch1蛋白的表达及TNF-α作用后Notch1蛋白的变化.结果 ①半定量RT-PCR检测结果表明Raji细胞经TNF-α作用后Notch1 mRNA的含量明显增高,且随TNF-α浓度的增加而增高,与对照组比较,差异有极显著性意义(P<0.01);②流式细胞术分析表明,Notch1蛋白在Raji细胞呈阳性表达,TNF-α作用组Notch1蛋白的平均荧光强度明显高于对照组,差异亦有极显著性意义 (P<0.01).结论 TNF-α可上调Raji细胞内Notch1蛋白的表达;TNF-α可通过影响Notch信号途径在淋巴瘤细胞内发挥作用.     

雷公藤内酯醇对非霍奇金淋巴瘤基质细胞衍生因子-1及其特异性受体生物学轴效应的影响

目的研究雷公藤内酯醇对非霍奇金淋巴瘤（non—Hodgkin lymphoma，NHL）细胞系Raji增殖的抑制作用，并探讨雷公藤内酯醇体外抑制NHL肿瘤细胞通过淋巴结转移的作用及其分子机制。方法采用MTT法测定雷公藤内酯醇对Raji细胞增殖的影响；采用RT—PCR方法检测雷公藤内酯醇对NHL淋巴结基质细胞中基质细胞衍生因子-1α（SDF—1α）表达的影响；采用流式细胞术检测雷公藤内酯醇作用前后NHL淋巴结瘤细胞CXCR4受体表达水平的变化；采用Transwell微孔隔离室迁移实验观察雷公藤内酯醇对NHL淋巴结瘤细胞体外迁移的影响。结果MTT法表明低剂量雷公藤内酯醇（6．25～25nmol／L）即可以时间和剂量依赖方式明显抑制Raji细胞的生长；其作用24、36、48、60、72h的IC50值分别为43．06、25．08、7．32、2．66、0．58nmol／L。RT—PCR结果显示雷公藤内酯醇能抑制NHL淋巴结基质细胞SDF-1α的表达，其中较低剂量组（12．5nmol／L）与对照组相比有一定的降低，但差异无显著性（P〉0．05），而较高剂量组（25、50nmol／L）则可明显抑制SDF-1α的表达（P〈0．05），并具有量效关系。流式细胞术分析结果发现经不同浓度雷公藤内酯醇处理后，NHL淋巴结瘤细胞CXCR4表达率与对照组相比逐渐下降（P〈0．01），此抑制效应呈剂量依赖性。Transwell趋化活性分析显示雷公藤内酯醇既能抑制重组人SDF-1α对NHL淋巴结瘤细胞的趋化作用，也能阻断NHL淋巴结基质细胞对瘤细胞的体外迁移，且雷公藤内酯醇对趋化作用的抑制具有剂量依赖关系。结论雷公藤内酯醇具有抗淋巴瘤细胞增殖的效应，并能阻断NHL淋巴结瘤细胞通过淋巴结的转移。其抗肿瘤机制及其抗细胞增殖作用与对NHLSDF-1／CXCR4生物学轴的抑制效应有关。     

Effects of Gambogic Acid on the Regulation of Nucleoporin Nup88 in U937 Cells

In order to investigate the anti-leukemia effects of gambogic acid （GA） and its relation to the regulation of nucleoporin Nup88 in U937 cells in vitro, the inhibitory effect of GA on the growth of U937 cells was examined by using MTT assay. Apoptosis was detected by Annexin-V FITC/PI double-labeled cytometry. Cell cycle regulation was studied by propidium iodide method. Both flow cytometry （FCM） and RT-PCR were employed to assess the expression of Nup88, and the localization of Nup88 was determined by confocal microscopy. The results indicated that GA had strong inhibitory effect on cell proliferation and apoptosis induction activity in U937 cells in vitro in a time-and dose-dependent manner. The 24-h IC50 value was （1.019±0.134） mg/L. Moreover, GA induced arrest of U937 cells in G0/G1 phase. Over-expression of Nup88 was found in U937 cells, whereas GA could significantly down-regulate both the protein and mRNA levels of Nup88. Nup88 was diffusely distributed between nucleus and cytoplasm and was located at the cytoplasmic side of nuclear rim, and occasionally in cytoplasm. It is suggested that GA exerts its anti-leukemia effects by regulating the expression and distribution of nucleoporin Nup88. It promises to be new agent for the treatment of acute leukemia.     

鱼藤素对U937细胞细胞周期及核孔蛋白Nup98和Nup88的调控作用

目的研究鱼藤素对人类白血病细胞株U937细胞体外抗肿瘤作用，研究其引起细胞周期与核孔蛋白Nup98和Nup88的改变，探讨其抗癌作用的分子机制。方法采用MTT法检测细胞增殖活性，流式细胞术检测细胞周期分布，激光共聚焦显微镜检测Nup98和Nup88蛋白表达变化，免疫电镜和流式细胞术观测鱼藤素对Nup98和Nup88的调控，Western blot检测鱼藤素对U937细胞Nup98和Nup88蛋白表达的影响。结果①鱼藤素对U937细胞具有明显的增殖抑制作用，其抑制作用呈时间和剂量依赖性。②鱼藤素作用U937细胞后细胞周期发生变化，0、5、10、20、40、80nmol／L鱼藤素处理U937细胞24h，主要使细胞阻滞于S期和G2／M期，而G1／G0期细胞呈浓度依赖性降低。G1／G0期细胞比例依次为73．01％、71．15％、68．42％、52．45％、43．99％和22．82％，S期细胞比例依次为17．18％、16．30％、18．09％、27．56％、31．21％和46．85％，G2／M期细胞比例依次为9．75％、12．31％、13．09％、18．99％、24．83％和27．79％。③鱼藤素可以调节U937细胞Nup98和Nup88的表达，低浓度即可使Nup98表达上调，而使Nup88表达下调。结论鱼藤素能够抑制U937细胞增殖，使细胞阻滞于S期和G2／M期，而G1/G0期细胞呈浓度依赖性降低。其抗肿瘤机制可能通过参与调控U937细胞Nup98和Nup88的表达，使Nup98表达上调，而使Nup88表达下调有关。     

蛋白酶体抑制剂MG132对姜黄素诱导K562和Raji细胞HDAC1、P300表达的研究

近年来的研究显示泛素-蛋白酶体通路在细胞组蛋白乙酰化、去乙酰化的调控中发挥了重要的作用.有研究报道姜黄素可抑制肿瘤细胞增殖,影响细胞周期,姜黄素可通过调节组蛋白乙酰化、去乙酰化而发挥其作用.我们通过探讨姜黄素和蛋白酶抑制剂MG132联含姜黄素作用K562细胞、Raji细胞对组蛋白乙酰化酶（HDAC1）、P300蛋白的影响进一步明确了姜黄素发挥作用是否是通过泛素-蛋白酶体通路.