H2O2对红细胞抗原性与LPO影响的FCM研究

用流式细胞计（flow cytometry,FCM）测定H₂O₂损伤后的红细胞（RBC）与IgG的结合能力,并直接检测细胞自发荧光的变化,以研究H₂O₂对RBC抗原性和脂质过氧化（LPO）荧光产物生成的影响.结果表明RBC抗原性和自发荧光变化与H₂O₂浓度和作用时间有关,抗原性变化对H₂O₂更敏感,比引起自发荧光明显增强所需H₂O₂浓度低两个数量级;还发现了RBC抗原性和自发荧光的变化与细胞的散射光有相关性.     

H2O2参与AM真菌与烟草共生过程

以烟草((Nicotiana tabacum,品种CF90NF)为寄主,苗期接种丛枝菌根(AM)真菌摩西球囊霉(Glomus mosseae,G.m),测定G.m与烟草共生过程中烟草根部H2O2含量以及多胺氧化酶(PAO)和过氧化物酶(POD)活性;研究外源H2O2对G.m侵染烟草的影响以及H2O2清除剂和合成抑制剂对烟草侧根H2O2含量及烟草侧根和菌丝中H2O2荧光强度的影响,以探究H2O2在AM真菌侵染烟草过程中的作用。结果表明,接种G.m 20d后烟草侧根中出现H2O2含量的猝发,一定浓度的外源H2O2促进G.m对烟草的侵染,而H2O2清除剂抗坏血酸(AsA)显著削弱烟草侧根和菌丝中的H2O2荧光强度,降低G.m对烟草的侵染率,表明H2O2参与G.m与烟草共生过程;在G.m与烟草共生过程中,PAO和POD活性显著升高,PAO抑制剂二氨基十二烷(DADD)和POD抑制剂水杨羟肟酸(SHAM)显著降低烟草侧根中H2O2荧光强度,对菌丝中H2O2荧光强度无显著影响,表明烟草根部和G.m均可产生H2O2,PAO和POD参与烟草侧根中H2O2的合成,菌丝中可能存在其他来源的H2O2。     

H2O2致体外培养动脉内皮细胞损伤及山莨菪碱保护

目的:实验以过氧化氢(H2O2)损伤体外培养动脉内皮细胞(AEC),对过氧化氢介导内皮细胞损伤、凋亡的机制进行探讨,观察山莨菪碱(Ani)是否抑制H2O2介导的AEC损伤、凋亡,并探讨Ani保护损伤、凋亡的机制.方法:体外培养AEC随机分组:对照组(正常培养AEC);H2O2损伤组(0.5 mmol/L H2O2损伤12 h);Ani组(不同浓度Ani:0.05,0.1,0.2 mg/mL处理45 min后加0.5 mmol/L H2O2损伤12 h).结果:1.低浓度H2O2(0.5 mmol/L)可以损伤AEC并诱导凋亡.H2O2损伤组台盼蓝着染率及LDH释放率均高于对照组(P<0.01);细胞总抗氧化能力(T-AOC)下降(比对照组P<0.01);琼脂糖凝胶电泳发现凋亡细胞的梯状电泳条带;细胞荧光染色见凋亡形态特征.2.Ani对H2O2诱导的内皮细胞损伤具保护作用.Ani组与H2O2损伤组比较,台盼蓝着染率及LDH释放率下降;T-AOC上升;在Ani 0.2 mg/mL组DNA琼脂糖凝胶电泳未见凋亡细胞特征性的梯状电泳条带;荧光染色未见凋亡细胞特征.结论:1.低浓度过氧化氢使AEC总抗氧化能力明显下降,耗竭细胞抗氧化能力,可致AEC的损伤、凋亡.2.山莨菪碱能减少AEC抗氧化能力的消耗,维持抗氧化能力平衡,保护0.5 mmol/L H2O2 介导的AEC损伤、凋亡.     

SB202190 调节蚕豆保卫细胞中SA 诱导H2O2 产生

运用激光共聚焦扫描技术, 在p38 MAP激酶专一抑制剂SB202190处理下, 探索植物促分裂原活化蛋白激酶(mitogenactivated protein kinase, MAP激酶)介导蚕豆(Vicia faba)保卫细胞中H2O2为代表的活性氧(reactive oxygen species, ROS)信号机制, 发现: p38 MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca2+探针荧光强度增强, 与水杨酸 (salicylic acid, SA) 或脱落酸 (abscisic acid, ABA) 迅速加强2种探针荧光强度形成鲜明对比; 而该抑制剂分别与SA和ABA共同处理, 前者H2O2探针荧光强度没有增加, 而后者荧光强度仍然能够增加; 而进一步使用Ca2+螯合剂BAPTA和SB202190 ＋SA共同处理, H2O2探针荧光强度没有增加。这些结果初步表明: 无论胞质Ca2+浓度高低, SB202190调节蚕豆保卫细胞中SA诱导H2O2产生, 但是不调节植物逆境信使分子ABA 此类的反应。因此推测, 植物细胞中可能有类似动物和酵母细胞中的p38MAP激酶类, 并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知, 这是首次报道SB202190和SA共同调节植物保卫细胞中ROS信号过程。     

异鼠李素抑制H2O2诱导的黑素细胞ROS水平升高和细胞活性降低

目的 探讨异鼠李素(isorhamnetin,ISO)对人表皮黑素细胞免受H2O2诱导氧化应激损伤的预保护作用.方法 不同浓度的H2O2处理人表皮黑素细胞,CCK-8检测细胞活性,筛选出H2O2体外诱导人黑素细胞氧化应激的最适浓度;用ISO预处理细胞后,使用最适浓度H2O2刺激,CCK-8检测细胞活性,NaOH裂解法测...     

AMP经p38MAPK修复H_2O_2致卫星细胞损伤机制分析

目的:探索AMP修复H2O2损伤骨骼肌卫星细胞作用机制。方法:取新生大鼠后肢肌肉传代培养获取卫星细胞,用各浓度AMP修复卫星细胞建立0.1mmol/L H2O2化学损伤模型;同时研究24h、48h、72h的AMP对损伤细胞保护,使用MTT与Giemsa染色,DAPI+FITC+PI荧光染色及免疫细胞荧光抗体检测。结果:各浓度AMP对卫星细胞作用不同,0.3125、0.15625mg/mL对细胞有损伤;2.5mg/mL AMP溶液对细胞增值和化学损伤修复最明显,5、1.25mg/mL AMP增值和化学损伤修复其次;以24h优于48h、72h,同时AMP修复细胞随时间延伸逐渐降低。其机制:(1)提高AMP/ATP比率激活AMPK蛋白调节细胞生物合成和增值;(2)增强凋亡抑制因子Bcl-2、抑制凋亡促进因子Bax。结论:适当浓度AMP对细胞能增值并经p38MAPK信号通路修复H2O2损伤卫星细胞。     

何首乌苷通过激活BDNF/TrkB信号通路拮抗H2O2诱导的大鼠海马神经元氧化损伤

探讨脑源性神经营养因子/酪氨酸激酶受体B(BDNF/TrkB)信号通路激活参与何首乌苷(PMG)对过氧化氢(H2O2)诱导神经元氧化应激损伤的保护作用。实验采用神经元原代培养,建立大鼠乳鼠海马神经元氧化应激损伤模型。实验结果显示高浓度的H2O2与MTT测定的细胞存活率降低相关,选择细胞存活率在40%~50%之间的200μmol/LH2O2浓度作为氧化应激损伤的实验浓度。与模型组相比,PMG预处理组(200μmol/L)可抑制H2O2诱导的神经元损伤(P<0.001)。TUNEL和β-微管蛋白III荧光染色显示PMG保护H2O2诱导的神经细胞损伤,明显降低细胞凋亡率(P<0.001),细胞骨架形态恢复正常。与PMG+H2O2预处理组相比较,当加入BDNF/TrkB信号转导通路阻断剂K252a后,PMG+H2O2+K252a组神经元细胞存活率大幅度下降(P<0.01),细胞骨架形态呈损伤状态。同时,我们发现PMG预处理恢复H2O2诱导的BDNF和P-TrkB的低表达水平,并且用K252a阻断BDNF/TrkB信号传导抑制了PMG对BDNF和P-TrkB表达水平的影响(P<0.01)。综上所述,何首乌苷可能通过激活BDNF/TrkB信号转导通路及维护神经元骨架的完整,实现对大鼠海马神经元氧化应激损伤的拮抗作用。     

高温和H2O2诱导酵母细胞产生活性衍生物的研究

对高温和H2O2应激条件下产生活性酵母细胞衍生物(Live Yeast Cell Derivative,简称LYCD)进行了研究。结果表明：低剂量的预处理(37℃和0．2mmol／LH2O2)能够增加细胞内谷胱甘肽(GSH)含量,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性。两种预处理均可以诱导对致死浓度H2O2的抗性。通过37℃和0．2mmol／LH2O2处理酵母细胞后,提取LYCD并添加到酵母细胞培养液中,发现细胞在致死浓度H2O2作用下的存活率明显提高,说明温度和H2O2刺激酵母细胞形成的LYCD对细胞氧化具有抵抗作用。     

Nrf2/HO-1信号通路在人诱导性多能干细胞氧化应激中的作用

氧化应激是诱导性多能干细胞(induced pluripotent stem cell, iPSC)在培养和应用中遇到的一个关键问题,探讨其作用机制具有重要的理论和实践意义。目前有关iPSC氧化应激的研究相对较少,Nrf2/HO-1信号通路在其中的作用尚不明了。因此,本研究以不同浓度的H2O2(100、200、300、400 μmol/L)处理人iPSC(hiPSC),分别在4 h和24 h于倒置显微镜下观察hiPSC及其饲养层细胞SNL氧化损伤的程度,通过碱性磷酸酶（alkaline phosphatase, AP）试剂盒和超氧化物阴离子荧光探针,分别检测hiPSC多能性和细胞活性氧(reactive oxygen species, ROS)水平,并通过qRT-PCR检测H2O2处理4 h后早期应激状态下Nrf2和HO 1 mRNA的表达水平,免疫细胞化学和Western印迹检测p-Nrf2和HO-1蛋白质的表达量。结果表明：hiPSC和SNL细胞的ROS水平呈H2O2剂量依赖性升高。除了100 μmol/L H2O2组hiPSC的细胞形态和多能性保持较好外,其余浓度H2O2均导致hiPSC出现不同程度损伤和死亡。但与SNL细胞相比,hiPSC中ROS水平相对较低,细胞状态也相对较好。SNL细胞中Nrf2和HO-1-mRNA表达的变化幅度与H2O2浓度呈线性相关,而hiPSC中Nrf2和HO-1表达的变化幅度与H2O2浓度之间并未呈现线性相关,其中Nrf2在100 μmol/L H2O2组表达量最高,而HO-1在200 μmol/L H2O2组表达量最高,意味着hiPSC氧化应激调控机制的复杂性。综上结果表明,hiPSC具有较好的抗氧化能力,其相关机制与Nrf2/HO-1信号通路有关,同时也可能涉及到其它相关通路的交互作用。     

活性氧不敏感型拟南芥的突变体对H2O2的响应

检测拟南芥ros突变株对H2O2响应的结果表明,此种突变体对H2O2有较强的耐受性,表现为气孔开度对H2O2不敏感和H2O2胁迫时的膜脂过氧化水平较低。采用激光扫描共聚焦显微术(LSCM)并结合H2O2荧光探针H2DCFDA检测外源ABA诱导保卫细胞的结果显示,突变体内荧光强度比野生型拟南芥低,暗示此种突变体消除H2O2的能力可能有提高,从而可增强植株抗氧化胁迫的能力。     

Modeling the adsorption of aromatic compounds on the TiO2/SiO2 catalyst

The Grand Canonical Monte Carlo method was used to analyse the phenomenon of adsorption of aromatic compounds (i.e. phenol, toluene, benzoic acid and salicylic acid) on the surface of the titania-silica (TiO₂/SiO₂) catalyst. We found that different types of interactions play important roles in the adsorption of molecules having polar and non-polar groups. Moreover, we found that the interactions between sorbate molecules are strong, and are the cause of multilayer adsorption occurring in the investigated temperature and pressure range. Presented at: Modeling and Design of Molecular Materials, 10–15 September 2006, Wrocław, Poland.     

Studies on the cytotoxic activity of synthetic 2H-azirine-2-azetidinone compounds

2-Azetidinones and 2H-azirines show antibacterial and cytotoxic activities, however the biological properties of molecules containing both 2H-azirine and 2-azetidinone functions in the same structure had never been evaluated before. In the present study, two 2H-azirine-2-azetidinones (1 and 2) and three 2H-azirines (3-5) were synthesized from 2-formyl-3-phenyl-2H-azirine-N-arylimines with diphenylketene. The compounds were assayed for antibacterial and cytotoxic activities. None of them showed antibacterial activity on the tested strains, but both 2H-azirine-2-azetidinones showed cytotoxicity against four tumor cell lines (HL-60, leukemia; HCT-8, colon cancer; MDA-MB-435, melanoma; and SF-295, CNS). The IC₅₀ values of 1 ranged from 1.1 to 10.5 μM and from 3.8 to 26.6 μM for 2. The mechanism of cell growth inhibition of 1 and 2 towards HL-60 cell line was also investigated. Membrane damage, cell viability, DNA synthesis inhibition and morphological changes were evaluated. The preliminary findings suggested that 1 and 2 induce apoptosis.     

Inhibitory and enhancing effects of NO on H2O2 toxicity: Dependence on the concentrations of NO and H2O2

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H₂O₂) toxicity and protect cells against H₂O₂ toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H₂O₂ toxicity in cultured liver endothelial cells over a wide range of NO and H₂O₂ concentrations. NO was generated by spermine NONOate (SpNO, 0.001–1 mM), H₂O₂ was generated continuously by glucose/glucose oxidase (GOD, 20–300 U/l), or added as a bolus (200 μM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H₂O₂-induced cell death. SpNO concentrations >0.1 mM were injurious with low H₂O₂ concentrations, but protective at high H₂O₂ concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H₂O₂ steady-state levels in line with inhibition of H₂O₂ degradation. Thus, the overall effect of NO on H₂O₂ toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H₂O₂ levels and enhancement being predominant with high NO and low H₂O₂ levels.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Prostaglandin E2 on the electroencephalogram

Present study was prompted by the report from another center on the occasional occurrence of convulsions and abnormal electroencephalogram (E.E.G.) patterns in women aborted with intraamniotic prostaglandin F2α (PGF2α). Fifty four subjects were investigated by means of an E.E.G. taken before and after initiation of PGE2 administration. They included pregnant and non-pregnant patients, nearly half (23) of whom were known epileptics. One seizure was observed during PG administration in a man with daily psychomotor attacks who had not taken his anticonvulsants on the day the test was performed. PGE2 caused no alteration of the E.E.G. in subjects with a normal control tracing; in those with an abnormal E.E.G., a deterioration was seen in one and an improvement in three. It is concluded that PGE2 is not epileptogenic at doses required for termination of pregnancy.