ADP-FeCl_3-黄嘌呤-黄嘌呤氧化酶体系产生的活性氧对脂质体的过氧化影响

ADP-FeCl_3-黄嘌呤-黄嘌呤氧化酶体系能引发卵磷脂脂质体的过氧化,但加入超氧化物歧化酶或过氧化氢酶能显著抑制该反应,而且两种酶的合并效应高于单独酶.根据这些结果,提出了ADP-FeCl_3-黄嘌呤-黄嘌呤氧化酶体系产生的活性氧对脂质体过氧化影响的机理.     

谷胱甘肽和半胱胺对黄嘌呤——黄嘌呤氧化酶反应生成的过氧化物所致细胞有丝分裂延迟的防护作用

照射时机体内产生多种自由基,使氧压增加,射线作用增强,因此在辐射损伤的产生过程中,氧起着重要作用。本文研究了谷胱甘肽(GSH)和半胱胺(MEA)对黄嘌呤一黄嘌呤氧化酶(X-XO)反应生成的O_2~-自由基引起细胞分裂延迟的防护作用。采用细胞培养法,测定含有20％小羊血清的伊格尔MEM培养液中L-5细胞分裂指数。X-XO反应中。O_2~-自由基的生成以nitroblutetrazolium     

晚期糖基化终产物诱导单核细胞产生细胞因子的细胞内信号传导机制

目的　探讨晚期糖基化终产物 (AGE)诱导单核细胞产生白细胞介素 1β(IL 1β)和肿瘤坏死因子 浕(TNF 浕)的细胞内信号传导机制。方法　采用密度梯度离心法分离健康成人外周血单核细胞 ,经AGE修饰的人血清白蛋白 (AGE HSA)刺激后 ,用ELISA法检测培养上清中IL 1β、TNF 浕水平 ,用化学发光法测定活性氧的生成 ;免疫细胞化学染色及凝胶迁移电泳 (EMSA)法观察核因子 κB(NF κB/p6 5 )的激活。分别用抗AGE受体 (RAGE)抗体、NADPH氧化酶特异性抑制剂apocynin和p38通路抑制剂SB 2 0 35 80预处理单核细胞 ,观察对AGE HSA诱导细胞因子和活性氧产生的影响。结果　AGE HSA与单核细胞在体外共同培养后 ,细胞内NF κB激活 ,培养上清中IL 1β、TNF 浕水平明显增高 ,活性氧生成增加 ;用抗RAGE抗血清或apocynin预处理细胞可阻断AGE HSA诱导的活性氧生成 (P <0 0 1) ,抑制NF κB的活化并使培养上清IL 1β、TNF 浕显著降低 (P <0 0 1)。用SB 2 0 35 80预处理细胞也可抑制AGE HSA诱导的NF κB激活及IL 1β、TNF 浕的产生 ,但对活性氧的产生无明显影响 (P >0 0 5 )。结论　AGE通过RAGE介导的途径刺激单核细胞生成IL 1β、TNF 浕和活性氧 ,NADPH氧化酶途径可能是RAGE细胞内信号途径的上游 ,而AGE诱导的细胞因子生成依赖于p     

葛根素对氧自由基的清除和抗氧化性损伤作用

目的　研究葛根素 (Puerarin ,Pue)对氧自由基的清除以及抗氧化性损伤作用。方法　以核黄素 -光系统产生超氧阴离子 ,Fenton反应产生羟自由基 (·OH) ,研究Pue对氧自由基的清除作用 ;以过氧化氢 (H2 O2 )引起红细胞溶血 ,黄嘌呤 -黄嘌呤氧化酶 (X -XOD)致豚鼠心室乳头肌损伤 ,研究Pue的抗氧化性损伤作用。结果　Pue能清除超氧阴离子和·OH ;抑制H2 O2 引起红细胞溶血和脂质过氧化物生成 ;增强心室乳头肌超氧化物歧化酶 (SOD)活性 ,降低丙二醛 (MDA)含量 ,保护乳头肌免受超氧阴离子的损伤。结论　Pue对氧自由基有清除作用 ,并能预防性对抗H2 O2 和超氧阴离子引起的氧化性损伤     

过度训练对大鼠腹膜巨噬细胞活性氧生成能力的影响及其相关机制研究

目的:了解过度训练对巨噬细胞活性氧生成能力的影响及其相关机制。方法:8周龄健康雄性Wistar大鼠16只,随机分为安静对照组和过度训练组,每组8只。过度训练组进行11周递增负荷跑台训练,最后一次训练结束后36 h断头处死,分离纯化腹膜巨噬细胞。流式细胞术测定其活性氧(ROS)生成量,荧光定量PCR技术测定NADPH氧化酶gp91phox、p22phox、p47phox亚基和葡萄糖-6-磷酸脱氢酶(G6PD)基因表达。结果:与安静对照组相比,过度运动组巨噬细胞ROS生成量显著降低(P=0.003),gp91phox、p22phox亚基表达量无显著变化(P>0.05),p47phox亚基表达量显著增加(P<0.05),G6PD表达量无显著差异(P>0.05)。结论:过度训练可显著降低大鼠腹膜巨噬细胞ROS生成水平,这并非通过抑制NADPH氧化酶亚基表达和抑制NADPH氧化酶生成途径实现,可能有其它调控机制参与。     

晚期氧化蛋白产物诱导HaCaT细胞凋亡的实验研究

目的 探讨晚期氧化蛋白产物(AOPPs)对HaCaT细胞凋亡的影响及其可能的作用机制.方法 人血清白蛋白(HSA)与次氯酸钠溶液反应制成AOPPs.MTT法检测经不同浓度(0、50、100、200、400μg/ml)AOPPs处理24h后HaCaT细胞的活力,以选择AOPPs的工作浓度.根据时间效应或浓度效应对HaCaT细胞进行分组处理,流式细胞技术检测细胞凋亡率,二氯荧光素双醋酸盐(DCFH-DA)法检测细胞内活性氧(ROS)水平,Western blotting技术检测细胞内NOX4、Bax及Bcl-2蛋白的表达.结果 400μg/ml AOPPs作用后HaCaT细胞活力较其他浓度明显降低(p＜o.05).随AOPPs作用时间的延长和浓度的增加,HaCaT细胞凋亡率明显增加(P＜0.05),细胞内ROS产生增多(P＜0.05),NOX4和Bax蛋白表达量增加(P＜0.05),Bcl-2蛋白表达量降低(P＜0.05).结论 AOPPs可能通过NADPH氧化酶途径诱导HaCaT细胞凋亡.     

赛庚啶对大鼠脑突触体Ca~(2+)水平和Ca~(2+)·Mg~(2+)-ATP酶活性的影响

观察赛庚啶(Cyp)对大鼠脑突触体Ca~(2+)水平和Ca~(2+)·Mg~(2+)-ATP酶活性的影响,以探讨其对脑缺血再灌注损伤的保护作用机理。用Quin2/AM法测定,突触体静息游离Ca~(2+)浓度为88±16μmol·g~(-1)蛋白,1.0,2.5和5.0μmol·L~(-1)的Cyp对其基本无影响。对高K~+所致突触体Ca~(2+)浓度升高有抑制作用但不显著,由185±45μmol·g~(-1)蛋白分别降至182±41、181±41、178±38μmol·g~(-1)蛋白;使突触体Ca~(2+)·Mg~(2+)-ATP酶活性由78±15mmol·h~(-1)·g~(-1)蛋白下降至74±12,70±11和65±8mmol·h~(-1)·g~(-1)蛋白。表明,Cyp是通过抑制Ca~(2+)内流和选择性抑制Ca~(2+)·Mg~(2+)-ATP酶活性,而稳定内质网和腺粒体内Ca~(2+)浓度,从而达到对脑缺血再灌注损伤的保护作用。     

胰酶处理牛乳中黄嘌呤氧化酶的提纯及其应用研究

用Massey法从胰酶处理牛乳中提纯黄嘌呤氧化酶,但对其程续进行某些改进.用改进程续纯化的黄嘌呤氧化酶的比活性为3.95IU/mg蛋白质而Massey报道的原始程续纯化的酶比活性为3.5IU/mg蛋白质.前者高于后者.纯化的黄嘌呤氧化酶已成功地应用于生物化学与自由基生物学和医学的研究.     

高热疗法促进5-氨基乙酰丙酸介导光动力疗法诱导胶质瘤细胞凋亡

目的:探讨光动力疗法(PDT)联合高热疗法(HT)的抗胶质瘤作用及可能的作用机制。方法:体外培养胶质瘤细胞株U87MG,共分为四组:对照组[HT(-)PDT(-)]、HT组[HT(+)PDT(-)]、PDT组[HT(-)PDT(+)]和PDT+HT组[HT(+)PDT(+)],分别给予相应处理;应用MTT实验评估细胞增殖能力;应用流式细胞术分析细胞凋亡率、线粒体膜电位和细胞内活性氧簇的生成情况;应用Western blot法检测细胞凋亡相关蛋白的表达情况。结果:MTT实验结果显示HT促进了PDT抑制U87MG细胞增殖,且与5-氨基乙酰丙酸浓度、超声辐照时间和HT温度呈正相关,并且伴随细胞活力下降的典型形态学变化。较低剂量(42℃作用10 min)的HT可明显促进PDT诱导细胞内活性氧生成增多、线粒体膜电位水平下降和细胞凋亡增加。此外,Western blot结果表明HT增进PDT上调了Caspase-3、-8、-9和Bax蛋白表达,下调了Bcl-2蛋白表达,而单独HT组却无明显变化。结论:HT可显著促进PDT抑制胶质瘤细胞增殖,促进细胞凋亡。其机制与上调Caspase-3、-8、-9和Bax,下调Bcl-2蛋白表达有关。     

血管紧张素-Ⅱ对肾上腺糖皮质激素生成的影响及其机制研究

本研究观察了血管紧张素-Ⅱ(A-Ⅱ)对豚鼠肾上腺皮质醇生成量的影响.当A-Ⅱ浓度≥10~(-9)M/L时,皮质醇的生成量显著增加(P<0.01),并且具有明显的量效关系和时效关系.A-Ⅱ和ACTH同时存在时,皮质醇的生成量明显高于A-Ⅱ和ACTH单独存在时的生成量(P分别<0.05).细胞外液Ca~(2+)缺乏时,A-Ⅱ促皮质醇分泌的作用有所降低,但未完全抑制(P<0.05).以上结果提示:高浓度A-Ⅱ对糖皮质激素的分泌具有直接的调节作用;A-Ⅱ和ACTH在这方面具有相互协同效应;Ca~(2+)参与A-Ⅱ促皮质醇分泌的机制.     

活性氧作用下肾上皮细胞的药物保护及机理

观察了三磷酸腺苷（ＡＴＰ）、过氧化氢酶（ＣＡＴ）及异搏停（ＶＥＲ）对活性氧作用下的肾上皮细胞（ＲＥＣ）的保护效果，以探讨其可能机制。结果显示预先应用ＡＴＰ、ＣＡＴ和ＶＥＲ均使处于活性氧作用下肾上皮细胞培养基中的乳酸脱氢酶（ＬＤＨ）活性及细胞活力维持或接近正常水准。提示能量危机、氧自由基（ＯＦＲ）毒性和钙超载可能是细胞缺血／再灌注（Ｉ／Ｒ）损伤病理生理中的重要环节，向细胞供能、清除ＯＦＲ及维持钙稳态是防治肾Ｉ／Ｒ损伤的重要措施。     

The formation and structure of the sulfoxyl radicals RSO(.), RSOO(.), RSO2(.), and RSO2OO(.) from the reaction of cysteine, glutathione and penicillamine thiyl radicals with molecular oxygen

This work reports an electron spin resonance study of the reactions of cysteine, glutathione and penicillamine thiyl radicals with molecular oxygen in frozen aqueous solutions at low temperatures. For all three thiols, the thiyl radical, RS., is found to react with oxygen to form the thiol peroxyl radical, RSOO(.). On the absorption of visible light, RSOO(.) photoisomerizes to the sulfonyl radical, RSO2(.), which subsequently reacts with molecular oxygen to form RSO2OO(.), the sulfonyl peroxyl radical. The identities of the sulfonyl and sulfonyl peroxyl radicals were confirmed by their production by a different route, from sulfinic acid. Sulfinyl radicals, RSO(.), are found as the final radical species in the reactions of thiyl radicals and oxygen. Parallel 17O hyperfine couplings (A parallel) are reported for each sulfoxyl radical and a correlation between the spin density on oxygen and the reactivity of the radical is suggested. As a result of this correlation sulfonyl peroxyl radicals are predicted to be far more reactive than thiol peroxyl radicals. We also report molecular orbital calculations on the nature of the spin density distribution and the molecular geometry of the model radicals CH3SO2(.) and CH3SO2OO(.).     

The influence of glutathione on single-strand breakage in single-stranded DNA irradiated in aqueous solution in the absence and presence of oxygen

The yields of strand break formation (Gssb) in single-stranded DNA (ssDNA) initiated by radiation-generated OH radicals have been determined using the method of low-angle laser light scattering (LALLS). The irradiations were carried out in aqueous, N2O-saturated solutions in the absence and presence of oxygen and at different concentrations of glutathione (GSH). GSH exhibits a protective effect, which is shown to be mainly due to OH radical scavenging. To quantify this, the rate constants for the reactions of OH with GSH and DNA have been redetermined under our experimental conditions. The values obtained were 9.0 x 10(9) and 4.5 x 10(8) dm3 mol-1 s-1, respectively. From the Gssb values obtained under anoxic conditions it is concluded that GSH protects against strand breakage (in addition to OH scavenging) by reacting with DNA radicals in competition to strand break formation. The rate constant of the repair reaction is 8.1 x 10(4) dm3 mol-1 s-1 at room temperature. For irradiations carried out in the presence of oxygen the rate of strand break formation is determined by the decay of DNA peroxyl radicals. Under these conditions we observed no protective effect of GSH apart from OH radical scavenging. The results are compatible with those that are expected from the oxygen-fixation hypothesis.     

PTEN基因敲除降低辐射敏感性及其机制

目的 探讨PTEN基因敲除对辐射敏感性的影响及机制。方法 采用流式细胞术检测MEF1和MEF1/PTEN^－/－细胞内ROS水平;采用Western blot方法,检测H₂O₂和DPI预处理后AKT激酶的表达变化;采用细胞克隆形成率试验分析细胞对⁶⁰Co γ射线的敏感性。结果 PTEN基因敲除后细胞ROS水平增加,辐射敏感性降低。H₂O₂和DPI预处理后影响MEF1细胞AKT激酶活性,但对MEF1/Pten^－/－细胞无影响。 结论 PTEN基因敲除阻断了ROS对AKT的介导,AKT激酶持续活化,可能是辐射敏感性降低的重要原因。     

抗辐灵活性成分对微波辐射致PC12细胞氧化应激损伤的保护作用研究

目的：观察抗辐灵活性成分黄芪总苷、赤芍总苷和丹参酮对微波辐射致PC12细胞氧化应激损伤的保护作用。方法采用神经生长因子（ nerve growth factor,NGF）诱导的PC12细胞为模型,分别给予1、3和9μg／ml黄芪总苷、赤芍总苷、丹参酮,作用48 h后经30 mW／cm2微波辐射6 min,于辐射后即刻采用倒置相差显微镜观察PC12细胞形态变化,四甲基偶氮唑盐（ methylthiazolyl tetrazolium,MTT）比色法测定细胞生存率,活性氧探针二氯二氢荧光素二乙酸酯（DCFH－DA）法测定细胞内活性氧（reactive oxygen species,ROS）水平,硫代巴比妥酸（thiobarbituric acid,TBA）法测定细胞脂质过氧化代谢产物丙二醛（ malonyldialdehyde,MDA）含量。结果3种活性成分作用后经微波辐射各组PC12细胞形态未见明显差异。辐射后6 h,辐射组PC12细胞较对照组（假辐射组）生存率明显降低（P＜0．01）;与辐射组相比,黄芪总苷（3μg／ml）给药组PC12细胞生存率明显提高（P＜0．01）。辐射后0和48 h,辐射组PC12细胞ROS、MDA水平明显升高（P＜0．01）,与辐射组相比,各药物预处理组PC12细胞ROS、MDA水平均显著降低（P＜0．05）。结论抗辐灵3种活性成分能改善微波辐射对PC12细胞的损伤,其保护作用可能是通过清除氧自由基和减轻氧化应激损伤来实现的。     

Effect of X-irradiation on erythrocyte membrane proteins. Primary radicals.

The effect of X-irradiation on the major proteins of human erythrocyte membranes have been examined. Samples of human erythrocyte ghosts and stripped ghosts were irradiated (up to 1.5 kGy) under air, N2 or N2O. The effects on the main erythrocyte membrane proteins as well as on aggregate formation were investigated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. Experiments were carried out with or without dithiothreitol as a reducing agent. The main peripheral protein of the erythrocyte membrane, spectrin, is more radio-sensitive than the other membrane proteins. Degradation was mainly due to aggregation and was increased by excluding oxygen. Since radiolysis under N2O instead of N2 enhanced the loss of spectrin, OH radicals seemed to be especially effective. Under anaerobic conditions the degradation of band 3 material could also be observed. In stripped erythrocyte ghosts the radiosensitivity of this integral protein was similar to that of spectrin.     

Fundamental model assays on chemical damages to ribonucleic acids caused by oxygen radicals in aqueous solution

A partial decomposition of RNA molecules in Saccharomyces cerevisiae is caused by photochemically produced superoxide anion radical (O2-.). Hydroxyl radicals (OH.) lead to complete destruction of RNA. Such radicals as originating from peroxidase-hydrogen peroxide reaction cause a partial decomposition of RNA molecules. All RNA species are damaged to the same extent by radicals. Damages due to oxygen radicals can be prevented or reduced by strong radical captors (dimethyl sulfoxide, 1,4-diazabicyclo-(2,2,2)-octane or superoxide dismutase). All these reactions proceed in aqueous solution. Correlations with the use of radicals in radiotherapy are discussed.     

Effects of 2-mercaptopropionyl Glycine on Radiation-induced Lipid Peroxidation in Liposomes and in Rat Liver Microsomal Suspensions

Summary

γ-Irradiation of rat liver microsomal suspensions resulted in the accumulation of both malondialdehyde (MDA) and lipid hydroperoxides. The presence of 2-mercaptopropionylglycine (MPG) during the irradiation period decreased the formation of MDA and lipid hydroperoxides in a dose (MPG)-dependent manner. This may be attributed to the ability of MPG to scavenge the free radicals produced by irradiation. Post-irradiation incubation of microsomes further enhanced the production of both MDA and lipid hydroperoxides; when high concentrations of MPG were present during the incubations the production of MDA and lipid hydroperoxides was substantially decreased. This antioxidant role of MPG was demonstrated for both pre-irradiated microsomes and liposomes and is thought to be due to the conversion of the hydroperoxy to hydroxy fatty acids within the lipid bilayer, as well as the scavenging action on initiating free radicals.     

The radiation hypersensitivity of cells at mitosis

PURPOSE: Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. MATERIALS AND METHODS: Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. RESULTS: Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. CONCLUSIONS: The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.     

Rate constants for the reactions of halogenated organic radicals

Absolute rate constants have been measured by means of pulse radiolysis for the reactions of various halogenated aliphatic compounds (ethane derivatives, including the anaesthetics halothane, enflurane, isoflurane and methoxyflurane) with hydrated electrons and .OH radicals, the reactions of halogenated carbon-centered radicals, derived thereby, with molecular oxygen, and the reactions of halogenated peroxyl radicals with various antioxidants (ascorbate, chlorpromazine, promethazine, propyl gallate, ABTS) in aqueous solutions. All oxygen addition reactions occur essentially diffusion-controlled. This finding is correlated with the stereoelectronic properties of the primary carbon-centred radicals. The oxidative power of the halogenated peroxyl radicals reflects the inductive -I effect of the halogens and accordingly increases with the degree of halogen substitution, with fluorine substituents being particularly effective. The peroxyl radicals derived from freon 113, namely CClF2CClFOO. and CCl2FCF2OO., have been identified as the best oxidants among these species.