黑加仑提取物保护血管内皮细胞氧化损伤的实验研究

目的研究黑加仑提取物对过氧化氢所致血管内皮细胞ECV-304损伤的影响。方法建立体外过氧化氢损伤模型,测定细胞存活率、上清液中丙二醛(MDA)含量、乳酸脱氢酶(LDH)活力、一氧化氮(NO)、内皮素(ET)和前列环素(PGI2)含量,并运用流式细胞仪观察黑加仑提取物对过氧化氢对内皮细胞ECV-304凋亡的影响。结果黑加仑提取物各剂量都能显著提高H2O2损伤内皮细胞的存活率(P<0.05);显著抑制H2O2损伤引起内皮细胞的LDH和ET的释放(P<0.05);显著减少H2O2损伤的内皮细胞MDA的生成(P<0.05);增加H2O2损伤的内皮细胞NO和PGI2水平(P<0.05)。黑加仑提取物的各组、阳性对照组和正常对照组早期凋亡率和总凋亡率明显低于H2O2模型组(P<0.05)。结论黑加仑提取物对过氧化氢损伤的脐静脉内皮细胞ECV-304具有保护作用;黑加仑提取物可降低H2O2诱导的ECV-304凋亡的早期凋亡率和总凋亡率,以达到保护内皮细胞的作用。     

亚硝酸钠对人胚肾细胞HEK-293毒性机制的研究

目的:探讨亚硝酸钠对人胚肾细胞HEK-293毒性机制。方法:将HEK-293人胚肾分别暴露于含0.0、3.0、6.0、12.0 mmol/L Na_2NO_2培养液中24 h, MTT法检测细胞活力,酶联免疫吸附法测定培养液中LDH、GGT、NAG、SOD、GSH、MDA含量。结果:低N组OD值、存活率高于对照组,中N组、高N组OD值、存活率低于对照组,差异有统计学意义(P0.01);随着Na_2NO_2剂量的增加,HEK-293 OD值、细胞存活率先升高(P0.01),然后明显降低(P0.01);低N组LDH、GGT、NAG水平低于对照组,中N组、高N组LDH、GGT、NAG高于对照组,差异有统计学意义(P0.01);随着Na_2NO_2剂量的增加,HEK-293 LDH、GGT、NAG先降低,后升高,剂量—效应关系明显(P0.01);低N组SOD、GSH高于对照组,MDA低于对照组,中N组、高N组SOD、GSH低于对照组,MDA高于对照组,差异有统计学意义(P0.01);随着Na_2NO_2剂量的增加,HEK-293 SOD、GSH先升高,后降低,MDA先降低,后升高,剂量—效应关系明显(P0.01)。结论:低剂量亚硝酸钠对HEK-293细胞为毒物兴奋效应,中高剂量亚硝酸钠对HEK-293细胞表现为抑制作用,其机制与细胞膜通透性改变、线粒体损害以及氧化损伤有关。     

叶酸与Vit B_(12)联合应用对糖尿病大鼠心肌细胞凋亡的调控

目的检测2型糖尿病大鼠血液中一氧化氮(NO)、肌酸激酶(CK)、超氧化物歧化酶(SOD)及微量丙二醛(MDA)的变化情况和心肌组织的凋亡情况,探讨叶酸和维生素B_(12)干预对大鼠心肌的保护作用。方法六周龄SD大鼠60只,糖尿病造模成功后,随机分为对照组(A组)、模型组(B组)、叶酸和维生素B_(12)干预组(C组)。12周后心脏取血,检测各组NO、CK、SOD及MDA的水平;取大鼠心肌组织,Tunel法观察心肌组织的细胞凋亡情况;免疫组化法检测Caspase-3、Caspase-8的表达,并用计算机图像分析系统测平均灰密度值。结果 12周后心脏血液中,B组NO、SOD水平较A组明显下降,C组较B组明显增高(P0.01);B组CK、MDA水平较A组明显增高,C组较B组明显下降(P0.01);心肌组织中,三组的凋亡指数分别为(2.6357±0.5214)、(16.2859±0.6983)、(4.0410±0.3121);Caspase-3各组分别为(79.2471±2.3547)、(97.1530±4.9522)、(84.1549±3.0198);Caspase-8各组分别为(70.2596±3.2118)、(98.9322±5.0583)、(78.3299±3.5680)。B组与C组细胞凋亡指数、Caspase-3、Caspase-8的表达均高于A组(P0.01),C组较B组明显下降(P0.01)。结论 2型糖尿病大鼠血液中NO、SOD、CK、MDA的变化参与心肌细胞的凋亡过程,而叶酸和维生素B_(12)的干预可以有效缓解心肌损伤。     

砷对大鼠生精细胞DNA损伤及XRCC1表达影响

目的 观察不同剂量三氧化二砷(As2O3)对成年大鼠生精细胞DNA损伤及其X射线修复交叉互补基因1(XRCC1)基因表达影响.方法 40只健康雄性SD大鼠随机分为4组,对照组、低、中、高剂量As2O3组(0.375、0.75、1.5 mg/kg),灌胃法连续给药16周处死大鼠,应用单细胞凝胶电泳试验检测大鼠生精细胞DNA损伤,免疫组化法检测大鼠生精细胞XRCC1蛋白表达.结果 对照组生精细胞平均尾长(1.04±0.61)μm,中、高剂量As2O3组可见部分细胞拖尾,平均尾长分别为(3.11±1.16)、(3.62±2.46)μm,明显长于对照组(P＜0.01),细胞尾部DNA含量比及尾矩也明显增加(P＜0.01);中、高剂量As2O3组XRCC1阳性细胞百分比分别为(11.13±7.06)％和(9.63±6.32)％,均较对照组的(15.49±8.23)％明显降低(P＜0.05),XRCC1表达量随着染毒剂量增高而降低;DNA损伤与XRCC1表达呈负相关(r=-0.778,P＜0.01).结论 一定剂量As2O3可通过抑制生精细胞XRCC1表达,诱导大鼠生精细胞DNA损伤,产生雄性生殖毒性.     

纳米硫化镉对人肾小球系膜细胞的氧化损伤研究

目的探讨人工合成纳米硫化镉对人肾小球系膜细胞(HMC)的损伤及可能的作用机制。方法采用不同浓度(0~80μg/ml)的纳米硫化镉对HMC细胞进行24 h染毒,采用MTT法检测纳米硫化镉对HMC细胞增殖的抑制作用,并选择0、10、20、30和40μg/ml纳米硫化镉剂量作用于HMC细胞,检测细胞内超氧化物歧化酶(SOD)及谷胱甘肽过氧化物还原酶(GSH-Px)活力、谷胱甘肽(GSH)及丙二醛(MDA)含量。结果与对照组相比,HMC细胞的存活率随着纳米硫化镉浓度的增加而下降(P0.05);纳米硫化镉染毒24 h后,40μg/ml组HMC细胞内的SOD及GSH-Px活力减弱(P0.05),GSH含量减少(P0.05),而MDA含量增加(P0.05)。结论抗氧化系统的损伤可能是纳米硫化镉引起HMC细胞毒性的机制之一。     

H2O2诱导建立HTR-8/SVneo胎盘滋养细胞氧化应激模型

目的通过观察不同浓度过氧化氢(H2O2)对人胎盘滋养层细胞HTR-8/SVneo处理不同时间后的氧化应激状态,探讨建立H2O2诱导HTR-8/SVneo胎盘滋养细胞氧化损伤模型的最佳条件。方法2017年11月至2018年2月于西安交通大学第一附属医院进行实验研究。将HTR-8/SVneo细胞实验组分别给予不同终浓度的H2O2(50、150、300、500μmol/L),同时设立对照组,相同实验条件下分别继续培养1、3、6、12h,随后对此进行细胞存活率、细胞形态学观察,对超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)、过氧化氢酶(CAT)测定,流式细胞仪分析活性氧自由基(ROS)水平和细胞凋亡的水平。结果与对照组比较,300μmol/L 3h经H2O2处理后可降低HTR-8/SVneo细胞的存活率(P=0.00<0.01),而且提高了LDH的释放,促使了SOD、CAT活性的下降和MDA含量的增加(P=0.00<0.01)。300μmol/L3h经H2O2处理后增加了细胞的凋亡率(P=0.00<0.01)及细胞内ROS水平(P=0.00<0.01)。同时,300μmol/L3h经H2O2处理后可明显促进细胞的形态学改变。结论通过H2O2可以成功建立HTR-8/SVneo氧化应激损伤细胞模型,最佳实验条件为300μmol/L H2O2处理3h。     

海参脑苷脂对H_2O_2致PC12细胞氧化损伤的保护作用

目的研究海参脑苷脂(SCC)对H2O2诱导PC12细胞氧化损伤的保护作用。方法建立H2O2致PC12细胞氧化损伤模型,MTT法检测细胞存活率;测定细胞培养液中乳酸脱氢酶(LDH)漏出量,评价细胞的损伤程度;利用荧光探针DCFH-DA对细胞内活性氧(ROS)进行荧光染色,检测荧光强度;化学比色法测定细胞内总抗氧化能力(T-AOC),黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性。结果海参脑苷脂能明显改善H2O2诱导的PC12细胞的氧化损伤,与模型组相比,SCC处理组可使细胞存活率升高,细胞培养液中LDH的漏出量明显减少(P<0.01),细胞内ROS的积累量明显降低(P<0.01),同时细胞内T-AOC和SOD活性明显增加(P<0.01)。结论 SCC对H2O2诱导的PC12细胞的氧化损伤具有一定的保护作用。     

适量运动和甲状腺激素对大鼠血液氧化应激反应的影响

目的 观察适量运动和甲状腺激素对大鼠血液H2O2含量和抗氧化能力的影响.方法 将64只Wistar大鼠随机分为8组:对照组(C)、轻度甲亢组(T1)、重度甲亢组(T2)、甲减组(J)、运动组(E)、轻度甲亢运动组(ET1)、重度甲亢运动组(ET2)、甲减运动组(EJ).C组生理盐水灌胃,T1和T2组分别给予左旋甲状腺素片100和200 μg/d灌胃,J组给予甲巯咪唑10 mg/d灌胃,运动为负重(6％体重)游泳训练30 min/d.喂养14d后,检测全血中的H2O2含量、谷胱甘肽过氧化酶(GPx)活力、红细胞超氧化物歧化酶(SOD)活力以及血浆丙二醛(MDA)含量.结果 与对照组比较,甲亢组H2O2含量、GPx活力、SOD活力和MDA含量均升高,差异有统计学意义(P＜0.05);甲减组H2O2含量、GPx活力、SOD活力和MDA含量均降低,差异有统计学意义(P＜0.05);与相应非运动组比较,运动组H2O2含量和GPx活力增高,SOD活力和MDA含量降低,差异有统计学意义(P＜0.05).结论 运动和甲亢均可诱发全血H2O2含量和抗氧化酶GPx活力增高,而且甲亢会诱发氧化损伤,但适量运动可减轻氧化损伤.大鼠血液对运动氧应激反应代偿作用强于甲亢时.     

邻苯二甲酸二乙基己基酯对大鼠睾丸细胞DNA损伤和睾丸组织氧化应激影响

目的 探讨邻苯二甲酸二乙基己基酯(DEHP)对大鼠睾丸细胞DNA的损伤和睾丸组织氧化应激的作用.方法 雄性Wistar大鼠25只,随机分为5组,每组5只,通过灌胃的方法摄入DEHP,4个染毒组剂量分别为1 500、3 000、4 500、6000mg·kg-1 ·d-1,1个阴性对照组.用单细胞凝胶电泳技术检测大鼠睾丸细胞DNA的损伤,睾丸组织中活性氧(ROS)、丙二醛(MDA)的水平以及睾丸组织中谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)的活力.结果 各染毒组和阴性对照组大鼠睾丸细胞DNA损伤率分别为29.0％、71.0％、89.0％、95.0％和15.0％,各染毒组睾丸细胞DNA损伤程度与对照组比较,差异均有统计学意义(P＜0.05,P＜0.01),且大鼠睾丸细胞DNA损伤率与染毒剂量之间呈剂量-反应关系(r =0.930,P＜0.01);各染毒组大鼠睾丸组织的ROS、MDA的水平均较阴性对照组显著升高(P＜0.05,P＜0.01),而GSH-Px、SOD的活力又较阴性对照组明显下降(P＜0.05,P＜0.01).结论 DEHP可造成大鼠睾丸组织明显的氧化应激并引起睾丸细胞DNA的损伤.     

葡萄籽原花青素对过氧化氢致大鼠海马神经元损伤的保护作用

[目的]观察葡萄籽原花青素（grape seed proanthocyanidin,GSP）对过氧化氢（H2O2）致大鼠海马神经元损伤的保护作用。[方法]实验分为5组：阴性对照组（A组）、H2O2模型组（B组）、GSP低剂量组（C组,1.25mg／L）、GSP中剂量组（D组,2.50mg／L）和GSP高剂量组（E组,5.00mg／L）。采用原代细胞培养的方法,建立H2O2致海马细胞氧化损伤模型。观察各组细胞形态和超微结构;采用四甲基偶氮唑盐（MTT）比色法检测细胞存活率;化学比色法检测细胞内超氧化物歧化酶（SOD）活性、活性氧（ROS）和丙二醛（MDA）冶量;TdT介导的dUTP缺口末端标记法（TUNEL法）检测细胞凋亡率。[结果]形态学观察C、D、E组均可减轻H2O2诱导的海马细胞氧化损伤。与B组相比,C、D、E组细胞存活率由31.97％分别上升到61.42％、68.47％和80.04％（P〈0.01）;细胞凋亡率由8.83％分别降低到3.50％、2.83％和2.00％（P〈0.01）;D、E组细胞内SOD含量明显增加（P〈0.05或P〈0.01）,C、D、E组抑制ROS能力和减少MDA含量明显增加（P〈0.01）。[结论]GSP对H2O2诱导的海马细胞氧化损伤有明显的保护作用,其作用机制可能与提高海马细胞的抗氧化能力以及减少凋亡率有关。     

Radiation-induced apoptosis is independent of caspase-8 but dependent on cytochrome c and the caspase-9 cascade in human leukemia HL60 cells

We investigated the role of the caspase activation cascade in apoptosis induced by ionizing radiation or hydrogen peroxide (H(2)O(2)) in human leukemia HL60 cells. Electron paramagnetic resonance (EPR) spectra revealed that hydroxyl and hydrogen radicals were generated in the culture medium after exposure to radiation or H(2)O(2). Initial accumulation of DNA fragments at 2 h after exposure was delayed in irradiated cells compared with H(2)O(2)-treated cells, although formation of abasic sites immediately after exposure was significantly higher in irradiated cells and similar quantities of hydroxyl radicals were produced under both conditions. Activity assay of caspases revealed that caspase-3, -8 and -9 were activated 2 h after exposure to H(2)O(2), whereas in irradiated cells caspase-3 and -9 activation occurred 4 h after exposure but increased caspase-8 activation was not observed. Release of cytochrome c into cytosol was seen at 2 h after radiation and H(2)O(2) treatment. Radiation did not affect proapoptotic proteins (Bax and Bid), whereas H (2)O(2) increased accumulation of Bax in the mitochondrial membrane 2 h to 6 h after treatment, independently of the truncation of Bid by activated caspase-8. Moreover, treatment with the caspase-8 inhibitor Z-IETD-FMK increased cell survival and prevented accumulation of DNA fragments in H(2)O(2)-treated cells, but not in irradiated cells. These results suggest that, unlike the caspase cascade of H(2)O(2)-induced apoptosis, cytochrome c and caspase-9 are important for the intrinsic pathway of radiation-induced apoptosis, independent of caspase-8.     

Mechanisms of the genotoxicity of crocidolite asbestos in mammalian cells: implication from mutation patterns induced by reactive oxygen species

Asbestos is an important environmental carcinogen in the United States and remains the primary occupational concern in many developing countries; however, the underlying mechanisms of its genotoxicity are not known. We showed previously that asbestos is a potent gene and chromosomal mutagen in mammalian cells and that it induces mostly multilocus deletions. Furthermore, reactive oxygen species (ROS) are associated with the mutagenic process. To evaluate the contribution of ROS to the mutagenicity of asbestos, we examined their generation, particularly hydrogen peroxide, and compared the types of mutants induced by crocidolite fibers with those generated by H(2)O(2 )in human-hamster hybrid (A(L)) cells. Using confocal scanning microscopy together with the radical probe 5,6 -chloromethy-2,7 -dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), we found that asbestos induces a dose-dependent increase in the level of ROS among fiber-treated A(L) cells, which is suppressed by concurrent treatment with dimethyl sulfoxide. Using N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent) together with horseradish peroxidase, we further demonstrated that there was a dose-dependent induction of H(2)O(2) in crocidolite-treated A(L) cells. The amount of H(2)O(2 )induced by asbestos reached a plateau at a dose of 6 microg/cm(2). Concurrent treatment with catalase (1,000 U/mL) inhibited this induction by 7- to 8-fold. Mutation spectrum analysis showed that the types of CD59(-) mutants induced by crocidolite fibers were similar to those induced by equitoxic doses of H(2)O(2). These results provide direct evidence that the mutagenicity of asbestos is mediated by ROS in mammalian cells.     

JWA基因参与细胞氧化应激的机制研究

目的　建立氧化应激模型 ,探讨新基因JWA参与细胞氧化应激的机制。方法　用H2 O2处理人乳腺癌细胞株 (MCF 7)和人胚胎肺纤维细胞株 (WI 38)两种细胞 ,观察细胞培养上清中丙二醛(MDA)和还原型谷胱甘肽 (GSH)的含量 ,用RT PCR半定量的方法及Westernblot蛋白质印迹法分别检测JWA在mRNA水平和蛋白质水平上的表达以及应激蛋白HSP2 7、HSP70、HSP90的表达。结果　MCF 7细胞培养上清中H2 O2 处理前MDA为 (0 .5 31± 0 .0 38)mmol/L ,处理后为 (0 .6 74± 0 .0 4 1)mmol/L ,差异有显著性 (P <0 .0 1) ;GSH在H2 O2 处理前为 (0 .0 5 3± 0 .0 0 2 )g/L ,处理后为 (0 .0 4 4±0 .0 0 2 )g/L ,差异有显著性 (P <0 .0 1)。WI 38细胞培养上清中H2 O2 处理前MDA的含量为 (0 .5 72±0 .0 35 )mmol/L ,处理后为 (0 .6 83± 0 .0 2 8)mmol/L ,差异有显著性 (P <0 .0 1) ;H2 O2 处理前GSH含量为(0 .0 5 8± 0 .0 0 2 )g/L ,处理后为 (0 .0 5 0± 0 .0 0 2 )g/L ,差异有显著性 (P <0 .0 1)。经H2 O2 孵育不同时间后 ,JWAmRNA在MCF 7细胞中明显下降 ,6h下降 6 8.4 % ,在WI 38细胞中则变化不明显 ;JWA蛋白和HSP2 7在两种细胞中的表达水平都有明显增加 ,但增加的程度不同。结论　JWA基因参与细胞氧化应激且在不同类型的细胞中     

Physiological concentrations of (-)-epigallocatechin-3-O-gallate (EGCg) prevent chromosomal damage induced by reactive oxygen species in WIL2-NS cells

We investigated the effects of (-)-epigallocatechin-3-O-gallate (EGCg) on chromosomal damage, which was evaluated by a cytokinesis-block micronucleus (CBMN) assay using WIL2-NS cells. EGCg itself induced chromosomal damage at 100 micromol/L. This damage was due to the production of H(2)O(2) by EGCg. In contrast, EGCg at < 10 micromol/L did not induce chromosomal damage and did not produce H(2)O(2). In addition, EGCg at < 10 micromol/L dose-dependently prevented chromosomal damage induced by H(2)O(2), tert-butyl hydroperoxide (tert-BuOOH) and superoxide, all of which are reactive oxygen species (ROS). A large amount of EGCg was present in cells after they were incubated with 0.3 micromol/L EGCg. When extracellular EGCg was removed and EGCg was present only inside of cells, the preventive effect of EGCg against tert-BuOOH-induced chromosomal damage was diminished but not that against the other two ROS tested. Direct interactions of EGCg with tert-BuOOH and superoxide but not with H(2)O(2) were detected. These findings suggest that physiological concentrations of EGCg (< 1 micromol/L) are not genotoxic but rather, can prevent ROS-induced chromosomal damage.     

砷对人淋巴细胞 DNA 氧化损伤的作用

目的：探讨砷（As)引起植物血凝集素（PHA）刺激和无刺激人外周血淋巴细胞DNA氧化性损伤。方法：用10μmol/L砷处理细胞2h,经单细胞凝胶电泳（SCGE,或彗星试验）－FPG（甲酰胺基嘧啶－DNA糖基化酶）消化法检测砷引起的DNA碱基损伤。结果：砷引起的DNA链断裂的修复过程与过氧化氢（H2O2）引起的修复过程类似,FPG消化产生的单链断裂,或砷引起的碱基损伤在PHA刺激淋巴细胞较未刺激细胞显著,在PHA刺激的淋巴细胞,砷和H2O2引起的DNA链断裂2h分别修复63％和68％,但在未刺激细胞分别修复大约34％和43％,在PHA刺激的淋巴细胞,砷和H2O2引起的碱基损伤2h分别修复40％和49％,但在未刺激细胞分别修复大约19％和21％。结果：微量砷可引起人类细胞DNA氧化性损伤,损伤的碱基主要是嘌呤或甲酰胺基嘧啶,未分裂（刺激）淋巴细胞修复砷与H2O2引起的DNA损伤较慢。     

Multi-Carotenoids at Physiological Levels Inhibit VEGF-Induced Tube Formation of Endothelial Cells and the Possible Mechanisms of Action Both In Vitro and Ex Vivo

Carotenoids have been shown to exhibit antiangiogenic activities. Several studies have indicated that carotenoids used in combination were more effective on antioxidation and anticancer actions than carotenoids used singly. However, it is unclear whether multi-carotenoids have antiangiogenic effects. We investigated the effects of multi-carotenoids at physiological plasma levels of Taiwanese (abbreviated as MCT, with a total of 1.4 μM) and Americans (abbreviated as MCA, with a total of 1.8 μM), and of post-supplemental plasma levels (abbreviated as HMC with a total of 3.55 μM) on vascular endothelial growth factor (VEGF)-induced tube formation in human umbilical vein endothelial cells (HUVECs) and rat aortic rings. MCT, MCA, and HMC inhibited VEGF-induced migration, invasion, and tube formation of HUVECs as well as new vessels formation in rat aortic rings. MCT, MCA, and HMC inhibited activities o\f matrix metalloproteinase (MMP)-2, urokinase plasminogen activator, and phosphorylation of VEGF receptor 2 induced by VEGF. Moreover, MCT, MCA, and HMC significantly upregulated protein expression of tissue inhibitors of MMP-2 and plasminogen activator inhibitor-1. These results demonstrate the antiangiogenic effect of multi-carotenoids both in vitro and ex vivo with possible mechanistic actions involving attenuation of VEGF receptor 2 phosphorylation and extracellular matrix degradation.     

Formamidopyrimidine-DNA glycosylase enhances arsenic-induced DNA strand breaks in PHA-stimulated and unstimulated human lymphocytes

To confirm that arsenic (As) induces oxidative DNA damage in phytohemagglutinin (PHA)-stimulated and unstimulated human lymphocytes, we used the alkaline comet assay combined with specific enzyme [formamidopyrimidine-DNA glycosylase (FPG)] digestion to measure As-induced base damage. The results showed that the enzyme-sensitive sites were readily detected with the alkaline comet assay after the cells were treated with 10 microM As for 2 hr. The repair patterns observed for FPG-created DNA single strand breaks (SSBs) in As-treated cells were comparable to those in hydrogen peroxide (H(2)O(2))-treated cells. The enzyme-created SSBs, As-induced base damage, were more significant in PHA-stimulated lymphocytes. About 63% and 68% of SSBs induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes by 2-hr repair incubation, but about 34% and 43%, respectively, were repaired in unstimulated cells. About 40% and 49% of base damage induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes, but about 19% and 21%, respectively, were repaired in unstimulated cells. These results indicated that As induced oxidative DNA damage in human lymphocytes at micromolar concentrations. The damaged bases could be chiefly purines or formamidopyrimidines. Like the damage induced by H(2)O(2), As-induced DNA damage was repaired more slowly in unstimulated lymphocytes.     

血红素加氧酶-1对H9c2心肌细胞氧化应激损伤的保护作用研究

目的:探讨血红素加氧酶-1(HO-1)对H9c2心肌细胞氧化应激损伤的保护作用及其作用机制。方法:建立过氧化氢(H2O2)诱导的H9c2心肌细胞氧化应激损伤模型,给予HO-1诱导剂氯化血红素(Hemin)预处理,或给予HO-1抑制剂锌原卟啉(Znpp-IX)共孵育。双波长法检测HO-1活性;四甲基偶氮唑盐(MTT)法检测细胞存活率;Hoechst33342染色和Caspase-3活性检测评估细胞凋亡;硫代巴比妥酸显色法检测丙二醛(MDA)含量,氮蓝四唑显色法检测总超氧化物歧化酶(SOD)活性;Western Blot法检测NF-κB蛋白表达水平。结果:与H2O2组相比,Hemin显著增加细胞HO-1活性,且该作用能被Znpp-IX阻断。与H2O2组相比,HO-1活性增加能够显著下调细胞凋亡率和Caspase-3活性,降低细胞MDA含量,增加总SOD活性,且上述作用能被Znpp-IX逆转。此外,HO-1活性增加能够显著抑制H2O2诱导的NF-κB激活,且该作用能被Znpp-IX逆转。结论:Hemin诱导的HO-1活性增加可能通过抑制NF-κB激活,维持细胞氧化还原平衡状态,抑制H2O2诱导的H9c2心肌细胞氧化应激损伤。     

过氧化氢处理K562细胞中JWA基因的表达及其与DNA损伤和凋亡的关系

目的研究过氧化氢(H2O2)诱导K562细胞氧化应激过程中,JWA蛋白、热休克蛋白(hsp70和hsp27)和p53蛋白的表达特点,探讨JWA参与细胞应答氧化应激的作用和可能机制。方法用0．01、0．1、1mmol／L的H2O2处理K562细胞10、30、60、180min建立氧化损伤模型;用0．1mmol／L H2O2处理不同时间(6～48h)和不同浓度(0．5～1000μmol／L)的H2O2处理48h分别诱导K562细胞凋亡,然后用DNA琼脂糖凝胶电泳鉴定DNA损伤和凋亡,用免疫印迹方法检测热休克蛋白(hsp70和hsp27)、p53以及JWA的蛋白表达水平。结果在DNA损伤模型中,H2O2活跃地调节JWA的表达,JWA对氧化应激的应答比热休克蛋白更快速,JWA和hsp70的表达规律相似;在低剂量H2O2(0．01mmol／L)处理时,JWA和热休克蛋白的表达均显著增高;在细胞凋亡模型中,JWA、hsp70、hsp27和p53的表达均显著增高,其中,JWA、hsp70和p53三者的表达规律相似。结论JWA是一个有效的环境应答基因并活跃地参与细胞应答氧化应激所致的DNA损伤和细胞凋亡的信号通路,其介导的信号通路可能与hsp70和p53有关。     

镁对人脐静脉内皮细胞DNA氧化损伤的影响

目的 :　观察 Mg2 + 对 H2 O2 诱导的人脐静脉内皮细胞 DNA氧化损伤的影响。方法 :　取新生儿脐带内皮细胞进行细胞培养。实验分四组 :1 .阴性对照。 2 .阳性对照 ,培养液中加H2 O2 。 3 .补镁组 ,培养液中加不同浓度的 Mg SO4 。 4.补 H2 O2 与镁组 :培养液中加 H2 O2 与镁。用单细胞凝胶电泳技术检测各组拖尾细胞率与拖尾细胞长。结果 :　与 H2 O2 组相比 ,H2 O2 +Mg2 + 各剂量组拖尾细胞率下降、拖尾细胞尾长减小。结论 :　 Mg2 + 对 H2 O2 诱导的 DNA氧化损伤有明显的拮抗作用。