PML(NLS~-)干扰对HL-60细胞增殖与凋亡的影响

目的探讨缺失核定位信号的PML,即PML(NLS-)干扰对急性早幼粒细胞白血病细胞HL-60增殖与凋亡的影响。方法将靶向PML(NLS-)基因的3组干扰质粒pGpu6-PML(NLS-)shRNA和阴性对照质粒pGpu6-NCshRNA分别转染HL-60细胞,转染后48 h,G418筛选阳性克隆,分别命名为Si-1、Si-2、Si-3和NC组,并设空白对照组。采用RT-PCR法和Western blot法检测各组细胞中PML(NLS-)基因mRNA的转录水平和蛋白的表达水平;MTT法检测细胞的增殖活力;流式细胞术分析细胞的细胞周期及凋亡情况。结果 Si-1和Si-2组HL-60细胞PML(NLS-)基因mRNA的转录水平和蛋白的表达水平与空白对照组相比明显减低(P<0.05),有干扰效果;Si-1组HL-60细胞的增殖水平与空白对照组相比明显降低(P<0.05),以转染后48 h降低最为显著(P<0.01);抑制PML(NLS-)表达可引起HL-60细胞S期比例增高,G1和G2期比例下降(P<0.05);Si-1组细胞凋亡率明显高于NC组和空白对照组(P<0.05)。结论干扰PML(NLS-)的表达可促进HL-60细胞的凋亡,抑制其增殖。     

NLS-RARα基因沉默对HL-60细胞增殖及分化的影响

目的探讨抑制NLS-RARα基因表达对急性早幼粒细胞白血病(Acute promyeolic leukemia,APL)细胞株HL-60增殖及分化的影响。方法将针对NLS-RARα基因的shRNA真核表达质粒(干扰组)和阴性对照质粒(阴性对照组)采用脂质体法转染HL-60细胞,并设未转染组,经G418筛选出稳定转染的细胞,采用MTT法检测细胞的增殖活力;RT-PCR和QRT-PCR法检测细胞中NLS-RARα基因mRNA的转录水平;Western blot法检测细胞中NLS-RARα蛋白的表达水平;流式细胞术检测细胞表面分化抗原CD11b的表达及细胞周期的分布。结果与阴性对照组和未转染组比较,干扰组HL-60细胞的增殖活力、NLS-RARα基因mRNA的转录水平和蛋白的表达水平均明显下降(P<0.05);CD11b的表达明显升高(P<0.05);G1、G2期细胞比例明显增加,S期细胞比例明显减少(P<0.05)。结论抑制NLS-RARα基因的表达可抑制HL-60细胞增殖,促进其分化。本实验为进一步研究APL发生发展的机制及APL的分子诊断和靶向治疗新途径奠定了基础。     

白藜芦醇诱导人皮肤鳞状细胞癌凋亡的蛋白质组学研究

为了探讨白藜芦醇抗皮肤鳞状细胞癌生物效应和分子机制,采用MTT法测定细胞活性,细胞流式法检测细胞凋亡率;SILAC蛋白质组学方法鉴定白藜芦醇调控的蛋白质分子。白藜芦醇呈浓度依赖性抑制皮肤鳞状细胞癌细胞的活性,细胞的存活率从100%降为56.7%;50μg/mL白藜芦醇处理导致37.3%细胞发生凋亡;鉴定了11个受白藜芦醇调控的蛋白质分子,其中BAG1,PDCD11,BCLAF1,HSPA9,YWHAZ等5个细胞凋亡相关蛋白被发现受白藜芦醇调控。     

流感疫苗诱导Hela细胞凋亡与免疫调节效应

目的观察流感疫苗诱导Hela细胞凋亡与免疫调节效应。方法将流感疫苗作用于Hela细胞,采用MTT比色法和流式细胞仪检测疫苗对Hela细胞增殖、凋亡和细胞周期的影响;同时用MTT法检测疫苗对小鼠脾细胞增殖的影响;采用结晶紫、中性红染色及MTT法,分别检测脾细胞诱导上清中IFN-γ、IL-2和TNF-α的分泌情况。结果一定浓度流感疫苗能够抑制Hela细胞增殖,促进细胞凋亡,凋亡率可达58·37%;还可促进小鼠脾细胞增殖及Th1型细胞分泌IFN-γ。结论流感疫苗能够抑制Hela细胞增殖,此作用可能是通过诱导Hela细胞凋亡、调节免疫细胞增殖及IFN-γ的分泌而实现的。     

紫杉醇诱导细胞凋亡的研究

目的探讨紫杉醇诱导细胞凋亡的效果。方法采用末端DNA片段原位标记法检测紫杉醇诱导细胞凋亡作用。结果正常SMC对照组偶见凋亡细胞,加入紫杉醇SMC,可见大量凋亡细胞。结论紫杉醇可诱导人脐动脉SMC的凋亡,其作用随药物剂量的增加而变强。     

姜黄素对人喉癌Hep-2细胞的生长抑制及诱导凋亡作用

目的探讨姜黄素(Curcumin)对人喉癌Hep-2细胞的生长抑制及诱导凋亡作用。方法将Hep-2细胞用含不同浓度姜黄素(3、6、12.5、25μmol/L)的培养基分别培养24和48h,采用MTT法检测其对细胞增殖的影响;台盼蓝染色法检测其对细胞活力的影响;流式细胞术检测其对细胞周期分布及细胞凋亡的影响;DNA琼脂糖凝胶电泳检测其对细胞DNA的影响。结果姜黄素可明显抑制Hep-2细胞增殖,且呈时间-剂量依赖性;可明显降低细胞活力,且呈剂量依赖性;可使细胞阻滞在G2/M期,并诱导细胞出现典型的凋亡峰,凋亡率呈时间-剂量依赖性;能使细胞DNA形成典型的DNA-Ladder。结论姜黄素对人喉癌Hep-2细胞增殖及细胞活力具有显著的抑制作用,并能诱导Hep-2细胞发生凋亡。     

人参皂苷Rh2诱导人胃癌SGC-7901细胞凋亡的研究

目的研究人参皂苷Rh2(G-Rh2)对人胃癌细胞SGC-7901的影响。方法采用MTT法检测G-Rh2对SGC-7901细胞存活率的影响;流式细胞分析法检测凋亡小体的含量;免疫印迹技术及体外Caspase-3/-7活力测定方法检测Caspase的激活状态。结果G-Rh2对SGC-7901细胞生长有明显的抑制作用,且呈量-效关系,IC50为9.3μg/ml。7.5μg/mlG-Rh2作用SGC-7901细胞24h,凋亡细胞数量为6.97%。7.5μg/mlG-Rh2作用SGC-7901细胞20h,出现多聚(ADP-核糖)聚合酶[poly(ADP-ribose)polymerase,PARP]断裂,Caspase-3/-7活力开始出现,并随作用时间的延长而增强。结论G-Rh2诱导Caspase参与SGC-7901细胞凋亡。     

腐植酸诱导塞尔托利细胞系-TM4生长迟缓

腐植酸是一种深褐色荧光的有机高分子复合物,它是由酚、醛、酸组成。在大鼠腹腔注射腐植酸能诱导大鼠睾丸形态的变化,包括曲细精管的退化,塞尔托利细胞和精原细胞数目的减少和精子细胞的丢失。有人认为塞尔托利细胞可能与睾丸萎缩的过程有关。在本研究中,使用了老鼠的塞尔托利细胞系-TM4研究腐植酸对塞尔托利细胞的影响和腐植酸诱导睾丸萎缩的机制。发现在与腐植酸接触的第1天至第4天TM4细胞生长减慢。使用流式细胞仪分析腐植酸处理的TM4细胞CDNA的含量,发现TM4细胞没有G1的次高峰,这表明TM4细胞对程序性细胞的死亡没有作用。然而,大部分的TM4细胞在G1期处于静止状态。在腐植酸处理4天后,处于G1期的TM4细胞的比例从36％增加到84％。腐植酸处理的TM4细胞的Western免疫印记分析表明：当p27^kip1蛋白的表达增加时细胞周期蛋白D1的表达在减少。研究结果表明,腐植酸诱导睾丸萎缩与对塞尔托利细胞的生长受到抑制有一定的关系。这个模型可能对环境试剂导致的睾丸萎缩的研究有作用。     

塞来昔布促进顺铂诱导人骨肉瘤细胞MG-63凋亡的作用

目的探讨塞来昔布促进顺铂诱导人骨肉瘤细胞MG-63凋亡的作用。方法将人骨肉瘤细胞分为塞来昔布组、顺铂组和联合组,分别加入不同浓度的塞来昔布、顺铂和塞来昔布+顺铂,采用MTT法检测细胞活性;流式细胞术检测细胞凋亡比例;吖啶橙/溴化乙锭(AO/EB)双染色观察细胞并计算凋亡率。结果塞来昔布单独作用于MG-63细胞后,细胞抑制率随药物浓度的升高而升高;联合组细胞抑制率均较顺铂组明显升高;FCM分析联合组细胞凋亡比例明显上升;AO/EB染色结果显示,联合组的细胞凋亡率明显高于塞来昔布组和顺铂组。结论塞来昔布和顺铂均可引起人骨肉瘤细胞的凋亡,两者联合使用可明显增强对人骨肉瘤细胞的凋亡作用。     

Kaempferol and Its Glycoside Derivatives as Modulators of Etoposide Activity in HL-60 Cells

Kaempferol is a polyphenol found in a variety of plants. Kaempferol exerts antitumor properties by affecting proliferation and apoptosis of cancer cells. We investigated whether kaempferol and its glycoside derivatives—kaempferol 3-O-[(6-O-E-caffeoyl)-β-D-glucopyranosyl-(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl-(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (P5) and kaempferol 3-O-[(6-O-E-feruloyl)-β-D-glucopyranosyl-(1→2)]-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (P7), isolated from aerial parts of Lens culinaris Medik.—affect the antitumor activity of etoposide in human promyelocytic leukemia (HL-60) cells. We analyzed the effect of kaempferol and its derivatives on cytotoxicity, DNA damage, apoptosis, cell cycle progression and free radicals induced by etoposide. We demonstrated that kaempferol increases the sensitivity of HL-60 cells to etoposide but does not affect apoptosis induced by this drug. Kaempferol also reduces the level of free radicals generated by etoposide. Unlike kaempferol, some of its derivatives reduce the apoptosis of HL-60 cells (P2 and P7) and increase the level of free radicals (P2 and P5) induced by etoposide. Our results indicate that kaempferol and its glycoside derivatives can modulate the activity of etoposide in HL-60 cells and affect its antitumor efficacy in this way. Kaempferol derivatives may have the opposite effect on the action of etoposide in HL-60 cells compared to kaempferol.     

N-acetylcysteine Can Induce Massive Oxidative Stress,Resulting in Cell Death with Apoptotic Features in Human Leukemia Cells

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O₂•⁻). Its subsequent conversion into H₂O₂ by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO⁻ from H₂O₂ by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O₂•⁻ in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.     

Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO₂-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.     

腐植酸在水产养殖中应用

本文概述了腐植酸在水产养殖中的作用机理和应用范围、效果,并展望了其发展前景,以期望为腐植酸的开发研究和推广应用提供帮助。     

腐植酸包涂尿素

介绍了腐植酸包涂尿素的工艺过程，经本工艺包涂的尿素样品，淋洗试验结果表明：氮的释放速率明显低于未包涂尿素氮的释放速率。     

腐植酸在改善生态环境中的应用

本文叙述了腐植酸特性及腐植酸类物质在改善生态环境中的应用。腐植酸可以有效地减少农药和化学肥料对生态环境的污染,净化养殖业的空气污染,能够治理工业及城市生活的污水、重金属和石油污染。因此,腐植酸类物质在改善生态环境中具有极其重要的作用,应用前景可观。     

腐植酸与不溶性腐植酸吸附铬的对比研究

通过对不溶性腐植酸和末处理的腐植酸对六价铬吸附作用的研究,确定了最佳反应条件,说明不溶性腐植酸的作用效果.试验表明,在反应接触时间60 min、酸性pH10左右的水溶液条件下,不溶性腐植酸对铬离子去除率可达98%.试验主要研究了不溶性腐植酸对铬离子的反应动力学和吸附等温线及其作用机理.     

电化学法处理水中腐殖酸的研究

以金属铝为阳极 ,采用电化学方法处理腐殖酸模拟废水 ,着重考察了溶液起始 pH值、电压、腐殖酸起始浓度与电解时间等因素对去除效果的影响。结果表明 ,在一定条件下牺牲阳极电解法可以十分有效地去除水体中的腐殖酸。腐殖酸的去除可能是·OH、H2 O2 氧化 ,直接电化学氧化和电凝聚共同作用的结果。     

土壤腐殖酸的提取及表征

从土壤中提取腐殖酸样品,并用物理和化学等方法对其进行了表征。同时,对提取过程中的一些问题如氮气保护、纯化也做了探索。结果表明有无氮气保护对最终产物无明显影响;ＨＦ－ＨＣｌ混酸能降低样品灰份;本腐殖酸含有的芳香结构较多,属大分子量低腐殖化的腐殖质级分     

用电渗析法分离煤中的黄腐酸研究

研究了一种新的黄腐酸提纯工艺方法 ,采用异相离子交换膜进行黄腐酸的电渗析提纯 ,黄腐酸经电渗析后的纯度可达 95% ,收率为 88.47% ;测试与分析表明 ,电渗析法对黄腐酸官能团结构无影响 ,电渗析试验未发生膜污染情况