Pten缺失小鼠胚胎成纤维细胞中活性氧和氧化损伤水平的研究

目的:研究Pten缺失后小鼠胚胎成纤维细胞抗氧化能力、细胞内活性氧(reactive oxygen species,ROS)水平、脂质以及DNA氧化损伤水平的变化.方法:采用超氧化物歧化酶(superoxide dismutase,SOD)活力检测、ROS荧光发光分析、丙二醛(malondialdehyde,MDA)舍量检测和γ-H2AX免疫荧光染色技术.分别比较Pten+/+ MEFs与Pten-/- MEFs细胞SOD活力、ROS、MDA和γ-H2AX水平的差异.结果:Pten-/- MEFs细胞中SOD活力明显减弱,ROS、MDA和γ-H2AX水平均显著增高.结论:Pten可能通过调控细胞抗氧化能力影响ROS水平,从而拮抗脂质和DNA氧化损伤以及由此产生的染色体不稳定性.     

Autophagy suppresses radiation damage by activating PARP-1 and attenuating reactive oxygen species in hepatoma cells

Abstract

Purpose: To investigate the relationship between autophagy and radiation damage of human hepatoma cells and to explore the role of reactive oxygen species (ROS).

Materials and methods: HepG2 cells were exposed to X-rays, then the protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and poly ADP-ribose polymerase-1 (PARP-1) were measured by Western blot assay, the formation of autophagosomes was detected by an autophagy detection kit, the intracellular ROS level was measured by flow cytometer, and DNA damage was evaluated by the incidence of micronuclei (MN). A CCK-8 kit was used to measure the proliferation ability of irradiated cells with or without N-acetyl-l-cysteine (NAC) treatment. In some experiments, the hepatoma cells were transferred with LC3 siRNA or PARP-1 siRNA before irradiation.

Results: The protein expressions of LC3 and PARP-1 and the inductions of autophagosomes and intracellular ROS were increased in the irradiated HepG2 cells. Pretreatment of cells with NAC relieved the irradiation-induced inhibition of cell proliferation. When HepG2 cells were transfected with the LC3 siRNA, the over-expression of PARP-1 was diminished in the irradiated cells. Compared with the control group, the inhibitions of LC3 and PARP-1 increased ROS level in the irradiated HepG2 cells and hence sensitized radiation responses of both proliferation inhibition and MN induction.

Conclusion: Autophagy upregulates the expression of PARP-1 and relieves radiation damage by reducing the generation of ROS.     

益肺活血颗粒对缺氧大鼠肺动脉平滑肌细胞内低氧诱导因子-1α和活性氧的影响

目的观察益肺活血颗粒对低氧培养大鼠肺动脉平滑肌细胞（pulmonaryarterysmoothmusclecells,PASMCs）内低氧诱导因子-1αhypoxiainduciblefactor一1alpha,HIF-1α和活性氧（Feacriveoxygenspecies,ROS）的影响。方法采用血清药理学方法制备不同浓度的益肺活血颗粒（yifeihuoxuegranule,YFHXG）含药血清,采用组织块贴壁法原代培养大鼠PASMCs,取对数生长期PASMCs随机分为常氧组、缺氧组、缺氧＋YFHXG组（16．5、3．3、0．66g／kg）。用噻唑蓝比色法测定各组PASMCs的增殖效应,免疫组化法测定细胞内HIF-1α蛋白的表达,激光共聚焦显微镜测定细胞内ROS的含量。结果与常氧组相比,缺氧组PASMCs增殖明显活跃,HIF-1α蛋白表达及ROS含量增加;与缺氧组相比,缺氧＋YFHXG高、中浓度组大鼠PASMCs的生长明显受抑制,而且HIF-1α蛋白表达及ROS含量降低。结论缺氧可以直接刺激PASMCs的增殖。YFHXG可以显著抑制低氧对大鼠PASMCs的促增殖作用,其机制可能是通过降低细胞内HIF-1α蛋白表达及ROS的水平来实现的。     

Kinetics of induction of reactive oxygen species during the post-irradiation expression of neoplastic transformation in vitro

Purpose : To examine whether the delayed expression of cell death and neoplastic foci in irradiated HeLa ×human skin fibroblast human hybrid cells correlates with the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells. Material and methods : HeLa ×human skin fibroblast human hybrid cells were irradiated and plated as for assay of neoplastic transformation. At regular time intervals during the post-irradiation expression period of 21 days, samples were harvested and assessed for the presence of oxyradical activity by measuring the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF) using flow cytometry. This reagent principally detects hydrogen peroxide. The kinetics of production of ROS were compared with those previously measured using the identical experimental protocol for the expression of both cell death and neoplastic foci. Results : The kinetics of production of ROS closely matched those for the onset of delayed cell death and delayed expression of neoplastic transformation. Conclusions : The induction of ROS is associated with delayed cell death and delayed neoplastic transformation in irradiated HeLa ×human skin fibroblast human hybrid cells.     

活性氧介导的铁过载对小鼠骨髓间充质干细胞的影响及其机制探讨

目的建立小鼠骨髓间充质干细胞(BM-MSCs)铁过载(IO)模型,并对铁过载模型小鼠进行去铁及抗氧化治疗,探讨铁过载对小鼠BM-MSCs的损伤作用及活性氧(ROS)在该损伤中的作用机制。方法采用随机区组设计,将40只雄性C57BL/6小鼠随机分为对照组、铁剂(右旋糖酐铁,25mg/ml)组(IO组)、铁剂+去铁治疗(DFX,125mg/kg)组(Fe+DFX组)、铁剂+抗氧化治疗(NAC,40mmol/L)组(Fe+NAC组),每组10只。从小鼠密质骨中分离BM-MSCs培养至P1代,检测BM-MSCs内铁颗粒、不稳定铁(LIP)及ROS水平;利用倍增时间及CCK-8试剂盒检测BM-MSCs增殖情况;采用碱性磷酸酶染色(ALP)、茜素红染色、成骨分化基因检测等方法评估BM-MSCs成骨分化能力;采用油红O染色检测BM-MSCs成脂定向分化能力。结果与对照组相比,铁剂组BM-MSCs内存在明显铁颗粒,LIP及ROS水平明显增高(P<0.05),倍增时间明显延长(2.07±0.14d vs 1.03±0.07d,P<0.01)。DFX组及NAC组倍增时间较铁剂组有所缩短,分别为1.52±0.07d与1.68±0.03d(P<0.05)。与对照组比较,铁剂组BM-MSCs矿化能力及向成骨细胞分化能力下降,成骨基因ALP、RUNX2、OSN表达增强,而成脂定向分化能力增强。在去铁及抗氧化治疗后,上述改变发生部分逆转。结论铁过载可影响小鼠骨髓MSCs的增殖及定向分化能力,其机制可能与铁过载所致ROS升高有关。     

Generation of reactive oxygen species after exhaustive aerobic and isometric exercise

Many studies have implicated elevated oxygen consumption (VO2) associated with aerobic exercise as contributing to oxidative stress. Only a few studies have investigated nonaerobic exercise and its relation to pro-oxidant and antioxidant activities. PURPOSE: The purpose of this study was to compare biomarkers of oxidative stress: lipid peroxidation, protein oxidation, and total antioxidants in blood after exhaustive aerobic (AE) and nonaerobic isometric exercise (IE). METHODS: Blood samples were collected from 12 subjects who performed a maximum AE and IE test and were analyzed for thiobarbituric acid (TBARS), carbonyls, lipid hydroperoxides (LH), and oxygen radical absorbance capacity (ORAC). RESULTS: VO2 increased 14-fold with AE compared with 2-fold with IE. Protein carbonyls increased 67% (P < 0.05) pre- to immediately and 1 h post-AE, and 12% pre- to immediately post-IE and returned to baseline 1 h post-IE. TBARS did not increase significantly with either treatment. LH increased 36% above rest during IE compared with 24% during AE (P < 0.05). ORAC increased 25% (P < 0.05) pre- to post-AE, compared with 9% (P < 0.05) pre- to post-IE. CONCLUSION: There was evidence of oxidative stress after both exhaustive aerobic and isometric exercise. Lipid hydroperoxides, protein carbonyls, and total antioxidants increased after both IE and AE. Due to the different metabolic demands of aerobic and isometric exercise, we can rule out a mass action effect of VO2 as the sole mechanism for exercise-induced oxidative stress.     

Kinetics of induction of reactive oxygen species during the post-irradiation expression of neoplastic transformation in vitro.

PURPOSE: To examine whether the delayed expression of cell death and neoplastic foci in irradiated HeLa x human skin fibroblast human hybrid cells correlates with the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells. MATERIAL AND METHODS: HeLa x human skin fibroblast human hybrid cells were irradiated and plated as for assay of neoplastic transformation. At regular time intervals during the post-irradiation expression period of 21 days, samples were harvested and assessed for the presence of oxyradical activity by measuring the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF) using flow cytometry. This reagent principally detects hydrogen peroxide. The kinetics of production of ROS were compared with those previously measured using the identical experimental protocol for the expression of both cell death and neoplastic foci. RESULTS: The kinetics of production of ROS closely matched those for the onset of delayed cell death and delayed expression of neoplastic transformation. CONCLUSIONS: The induction of ROS is associated with delayed cell death and delayed neoplastic transformation in irradiated HeLa x human skin fibroblast human hybrid cells.     

亚低温对于心肺复苏后大鼠海马神经细胞ROS产生量和Caspase-3mRNA表达的影响

目的观察亚低温（Hypothermia,HT）对心肺复苏（Cardiopulmonaryresuscitation,CPR）后大鼠海马神经细胞活性氧（Reactiveoxygenspecies,ROS）产生量和半胱氨酸天冬氨酸蛋白酶3（Caspase-3）mRNA表达的影响。方法37只健康成年雄性SD大鼠随机分为3组：空白对照组、常温CPR组、亚低温CPR组。CPR组再分2个亚组：即自主循环恢复（ReturnofSpontaneousCirculation,ROSC）后12h、24h组,除空白对照组为5只大鼠外,余各亚组大鼠均为8只。亚低温CPR组在ROSC后立即行亚低温干预。到达各观察时相点时立即取材,应用流式法检测大鼠海马单细胞悬液中活性氧水平,逆转录-聚合酶链反应（RT—PCR）技术测定海马神经细胞Caspase-3mRNA的表达水平。结果亚低温CPR组ROSC后12h、24h的ROS产生量和Caspase-3 mRNA表达与相同时相点常温CPR组比较均显著降低（P〈0．05）。结论HT能减少CPR后大鼠神经细胞ROS的产生,并能通过抑制Caspase-3mRNA表达而使神经细胞凋亡减少。