离体成纤维细胞热损伤模型制作的研究

目的 建立离体细胞烫伤诱导凋亡的模型，深入研究组织烫伤后主要修复细胞成纤维细胞的变化。方法 利用体外细胞培养技术对含小牛血清（体积分数为５％和10％）的DMEM培养液中的成纤维细胞进行不同温度（43，45，48℃）和不同时间（10,30,40min）的处理，对照组细胞置于37℃水浴30min。采用DNA凝胶电泳、Hoeschst33258荧光染色、逶射电镜等技术对烫伤细胞进行检测并分析结果。结果 在水浴中，成纤维细胞育10，30，40min均可出现细胞凋亡，以含体积分数为5％小牛血清的DMEM培养液、45℃水浴10min一组最为明显。48℃中水浴30min以上，则出现大量的细胞坏死。对照组中无明显的细胞凋亡发生。结论 热损伤能造成离体成纤维细胞发生凋亡。该方法有助于在离体情况下探讨创伤修得过程中成纤维细胞生物学特性。     

碱性成纤维细胞生长因子对脊髓损伤后神经再生的影响

脊髓损伤后功能恢复不良的主要原因为伤后继发性损害不断加重 ,发展成为不可逆损害。如何保护脊髓组织、减少脊髓继发性损害成为当今研究的重点。碱性成纤维细胞生长因子 (ｂａｓｉｃｆｉ ｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ)是近年来引起人们关注的新的神经营养因子 ,在机体的胚胎发育、血管形成、创伤愈合及神经系统生长发育过程中起重要作用[1 ] 。为探讨ｂＦＧＦ对脊髓损伤后神经组织再生的影响 ,笔者通过免疫组织化学方法及特殊染色手段 ,观测ｂＦＧＦ对脊髓损伤后神经再生的影响 ,为临床应用ｂＦＧＦ治疗脊髓损伤提供…     

核因子κB降低小鼠胚胎成纤维细胞中Lmp2基因转录

目的 研究在小鼠胚胎成纤维细胞(MEF)中,核因子κB(NF-κB)对Lmp2基因转录的影响.方法 利用NF-κB荧光素酶报告基因检测MEF和c-abl/arg缺失株(DKO)中内源性NF-κB活性;构建包含Lmp2启动子序列和NF-κB结合序列突变体的荧光素酶报告基因,通过双荧光素酶报告系统,研究NF-κB对Lmp2启动子转录活性的调控,并应用定量PCR、Western印迹比较Lmp2基因在MEF和DKO细胞中表达水平.结果 DKO细胞中内源性NF-κB活性上调;DKO细胞中Lmp2基因启动子的转录活性发生下调,并且NF-κB结合序列突变后,其转录活性升高;定量PCR和Western印迹结果表明,DKO细胞中Lmp2基因mRNA和蛋白表达水平比较MEF细胞均发生下调.结论 MEF中内源性NF-κB可负调控Lmp2基因转录,并降低其蛋白表达水平.     

褪黑素对大鼠实验性脊髓损伤的神经保护作用

目的观察不同剂量褪黑素对实验性脊髓损伤(SCI)大鼠早期脂质过氧化水平及超微结构的神经保护作用。方法采用钳夹法在大鼠T10水平建立SCI模型。60只SCI模型大鼠随机分为6组:模型Ⅰ组仅行椎板切除,模型Ⅱ组行椎板切除+钳夹,对照组损伤后立即给予5%无水乙醇0.2ml,甲泼尼龙治疗组给予甲泼尼龙30mg/kg,褪黑素治疗Ⅰ组给予褪黑素50mg/kg,褪黑素治疗Ⅱ组给予褪黑素100mg/kg。于伤后24h检测各组大鼠受损部位脊髓丙二醛(MDA)含量并进行超微结构病理改变评分。结果模型Ⅰ组及各治疗组MDA含量明显低于模型Ⅱ组和对照组(P<0.05),褪黑素治疗组与甲泼尼龙治疗组间差异无统计学意义(P>0.05),两个褪黑素治疗组之间差异亦无统计学意义(P>0.05)。超微组织病理改变评分结果显示,褪黑素治疗Ⅱ组的神经保护作用与甲泼尼龙治疗组相当(P>0.05),褪黑素治疗Ⅱ组的结果优于褪黑素治疗Ⅰ组(P<0.05)。结论褪黑素可抑制实验性脊髓损伤早期脂质过氧化水平,从而发挥神经保护作用。     

离体热损伤成纤维细胞所涉及的细胞信号通路

利用已建立的成纤维细胞热损伤诱导凋亡模型观察离体培养的成纤维细胞热烫伤后信号通路的活化情况。首先将原代培养的人成纤维细胞经5～6传代后，45℃水中孵育10min，造成单纯热损伤刺激，加入5％小牛血清继续培养，分别在伤后0、30、60和180min 4个时相点冻存细胞，运用Western blot进行MAPKs信号通路磷酸化蛋白印迹检测，同时采用荧光显微镜检测热损伤成纤维细胞caspase3蛋白的表达情况。结果显示，经过热损伤刺激的成纤维细胞，c-jun N末端激酶(JNK)首先开始表达，并在60min达到高峰，能持续至180min。细胞外信号调节激酶(ERK)的表达高峰在伤后30min，随后迅速消退。损伤早期，成纤维细胞内caspase3阳性标记的细胞数较少，至伤后60min，caspase3的表达明显增强。研究表明，ERK和JNK信号通路在热损伤诱导成纤维细胞生物学特性变化过程中具有重要意义。     

小鼠胚胎成纤维细胞复制性衰老模型的构建及衰老溶酶体功能检测

目的 构建小鼠胚胎成纤维细胞（MEF）复制性衰老模型,检测衰老溶酶体相关功能改变,为衰老相关溶酶体疾病提供有效的细胞模型。方法 通过酶消化法分离提取MEF,体外连续传代培养构建MEF复制性衰老模型,实时定量PCR(qPCR)检测衰老标志物p16和p21,衰老相关β-半乳糖苷酶（SA-β-gal）染色方法进一步验证衰老状态;通过溶酶体探针类染料LysoTracker Red DND-99探针及LysoSensor Yellow/Blue DND-160双激发探针追踪酸性溶酶体定性检测溶酶体酸碱性及定量检测溶酶体pH值;通过偶联自淬BODIPY?染料的牛血清白蛋白（DQ-BSA）检测衰老溶酶体的降解能力。结果 qPCR和SA-β-gal染色结果显示,MEF复制性衰老模型构建成功。与年轻P3代MEF相比,衰老P9代MEF溶酶体的酸性明显丧失,差异有统计学意义（P<0.0001）,衰老P9代MEF溶酶体的降解能力明显降低（P<0.05）。结论 成功构建MEF复制性衰老细胞模型,并证明衰老MEF溶酶体的酸性丧失以及降解能力下降。     

微波辐照对小鼠肾皮质与睾丸中MDA含量和SOD活性的影响

目的：研究微波辐照对小鼠肾皮质和睾丸中脂质过氧化产物丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性的影响。方法：选用频率2450MHz、功率密度10mW/cm^2的微波照射小鼠,用TBA比色法和NBT羟胺法,检测组织中MDA含量和SOD活性。结果：微波辐照后1,6,12,24d,小鼠的肾皮质和睾丸中MDA含量升高,24d达到最高量（P＜0．01）;SOD活性均降低,24d降到最低点（P＜0．01）。结论：该辐照条件下,微波辐照可致肾皮质和睾丸中自由基产量过多,SOD活性降低。     

兔视网膜氪激光损伤修复中碱性成纤维细胞生长因子和Ⅰ/Ⅲ型胶原基因的表达变化

目的：探讨碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF)和I／Ⅲ型胶原基因与视网膜激光损伤后修复的关系。方法：用氪激光致伤有色家兔后极部视网膜，于伤后1，3，7，14d取眼球做冰冻切片，用组织原位杂交法观察bFGF mRNA和Ⅰ/Ⅲ型胶原纤维mRNA表达的变化。结果：光凝后1－3d，邻近光凝斑的兔视网膜节细胞层和内核层中bFGF mRNA阳性表达明显增加，在相同部位和时相Ⅰ/Ⅲ型胶原纤维mRNA亦呈上调表达。结论：Ⅰ/Ⅲ型胶原基因与视网膜损伤修复直接相关，bFGF可能起重要的调节作用。     

磁刺激和碱性成纤维细胞生长因了对脊髓损伤后神经再生的影响

脊髓损伤预后不良的主要原因为伤后继发性损害和神经再生困难。碱性成纤维细胞生长因子（BFGF）是一种能广泛促进来源于中胚层及神经外胚层细胞增殖的多肽生长因子，对神经具有保护作用。     

Pten基因敲除对过氧化物酶家族表达和活性氧水平的影响

目的:探讨Pten基因敲除后对过氧化物酶家族(Peroxiredoxins,Prdxs)水平和活性氧水平的影响.方法:采用Western印迹和化学/荧光发光分析法分别检测了在Pten / MEF和Pten-/-MEF细胞中PRDXs的表达和细胞内活性氧水平.结果:Western印迹结果显示,与Pten / MEF细胞相比,Pten-/-MEF细胞PRDX Ⅰ,Ⅱ,Ⅴ,Ⅵ蛋白水平下调,PRDX Ⅲ不变,PRDX Ⅳ上调.DCFH探针标记后流式结果显示Pten-/-MEF 细胞活性氧荧光值显著高于对照Pten / MEF细胞(P＜0.05).结论:Pten基因敲除引起数种PRDXs表达下调,细胞内活性氧水平增高.     

Effects of dietary carbohydrate on delayed onset muscle soreness and reactive oxygen species after contraction induced muscle damage

Background: Delayed onset muscle soreness (DOMS) occurs after unaccustomed exercise and has been suggested to be attributable to reactive oxygen species (ROS). Previous studies have shown increased ROS after lengthening contractions, attributable to invading phagocytes. Plasma glucose is a vital fuel for phagocytes, therefore carbohydrate (CHO) status before exercise may influence ROS production and DOMS Objective: To examine the effect of pre-exercise CHO status on DOMS, ROS production, and muscle function after contraction induced muscle damage. Method: Twelve subjects performed two downhill runs, one after a high CHO diet and one after a low CHO diet. Blood samples were drawn for analysis of malondialdehyde, total glutathione, creatine kinase, non-esterified fatty acids, lactate, glucose, and leucocytes. DOMS and muscle function were assessed daily. Results: The high CHO diet resulted in higher respiratory exchange ratio and lactate concentrations than the low CHO diet before exercise. The low CHO diet resulted in higher non-esterified fatty acid concentrations before exercise. DOMS developed after exercise and remained for up to 96 hours, after both diets. A biphasic response in creatine kinase occurred after both diets at 24 and 96 hours after exercise. Malondialdehyde had increased 72 hours after exercise after both diets, and muscle function was attenuated up to this time. Conclusions: Downhill running resulted in increased ROS production and ratings of DOMS and secondary increases in muscle damage. CHO status before exercise had no effect.     

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.     

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.     

Nitric oxide, reactive oxygen species, and skeletal muscle contraction

Nitric oxide (NO) derivatives and reactive oxygen species (ROS) modulate contractile function of respiratory and limb skeletal muscle. The intracellular processes regulated by NO and ROS remain enigmatic, however. Studies of reduced preparations have identified a number of regulatory proteins that exhibit altered function when exposed to exogenous NO or ROS donors ex vivo. The relative importance of these targets in the intact cell is not known and conflicting theories abound regarding the mechanism(s) whereby NO and ROS regulate contraction. This review article provides a personal perspective on the processes regulated by NO and ROS by addressing three major topics: 1) the regulatory mechanisms by which endogenous NO depresses force production, 2) the processes whereby endogenous ROS modulate contraction of unfatigued muscle, and 3) the site(s) of action and reversibility of ROS effects in muscle fatigue.     

高压氧对人血淋巴细胞DNA氧化损伤的影响

目的　探讨高压氧（ＨＢＯ）治疗对人体血液淋巴细胞ＤＮＡ氧化损伤的影响。方法　１６ 名健康男性志愿者随机分为甲、乙两组,均置于ＨＢＯ（０．２５ ＭＰａ）治疗环境中,每天暴露２０ 分钟３次,中间吸空气５分钟,共３ 天。甲组单纯ＨＢＯ处理,乙组在ＨＢＯ暴露前三天开始静脉滴注维生素Ｃ１ ｇ,每日１ 次。分别于第一天ＨＢＯ暴露前和暴露后即刻、１２ 小时、２４ 小时,第２ 天和第３ 天ＨＢＯ暴露前和暴露后即刻采集静脉血样,经单细胞凝胶电泳分析结合ＦＰＧ定量检测ＤＮＡ碱基的氧化损害。结果　第一天ＨＢＯ 暴露后即刻、１２ 小时较暴露前明显存在氧化损害（Ｐ＜ ０．０１）,第一天ＨＢＯ 后２４ 小时未见明显ＤＮＡ氧化损害（Ｐ＞ ０．０５）;第２天和第３天ＨＢＯ暴露后即刻未见明显ＤＮＡ 氧化损害（Ｐ＞ ０．０５）;预先注射维生素Ｃ的乙组第一天ＨＢＯ暴露前、后均未见明显ＤＮＡ 氧化损害（Ｐ＞ ０．０５）。结论　ＨＢＯ治疗后立即对ＤＮＡ碱基产生氧化损害;２４小时内机体抗氧化防御系统能力提高,ＤＮＡ 碱基氧化损害消失;抗氧化剂维生素Ｃ可以防止这种损害。     

Does hydrostatic pressure have an effect on reactive oxygen species in the eel?

Eels are submitted to hydrostatic pressure (HP) during their spawning migration (about 6000 Km). Before migration, they change from the yellow to the silver stage (silvering process). The effects of HP in relation to the silvering process have been studied on aerobic metabolism and more precisely on reactive oxygen species (ROS) metabolism. HP acclimatization of yellow eels improves oxidative phosphorylation together with supposed concomitant changes in electron leak and ROS production. Therefore hydroxyl radical (OH*) production, superoxyde dismutase and catalase activities, malondialdehyde content and in parallel oxygen consumption were measured in the red muscle of long-term pressure exposed and control group yellow and silver eels. At atmospheric pressure, yellow eels exhibited significantly higher oxygen consumption and OH* production than silver eels; and significantly lower malondialdehyde content. This could be due to the increase in membrane fluidity induced by the silvering process. Long-term HP exposure decreases yellow eel oxygen consumption which becomes similar to that of the silver stage. In parallel there is a decrease in OH* production and concomitantly antioxidant enzyme activities follow the same tendency. Thus the respiratory chain improvement in pressure acclimatized yellow eels is accompanied by a ROS production decrease which could mean an electron leak decrease.     

LeuCAM and reactive oxygen species during long-term exercise combined with sleep and energy deficiency

INTRODUCTION: During the Norwegian military ranger-training course, cadets are exposed to prolonged physical exercise combined with sleep-, energy-, and food deficiency. The open-window postexercise hypothesis indicates that after hard physical activity, there is an increased risk of contracting infectious diseases. PURPOSE: The purpose of the present study was to determine leukocyte reactive oxygen species (ROS) levels, total antioxidant status (TAS), leukocyte expression of the cell adhesion molecules CD62L and CD11b, and plasma levels of soluble adhesion molecule L-selectin before, during, and in the recovery phase of a military ranger-training course. METHODS: Ten cadets from the Norwegian Military Academy were recruited to the study. Flow cytometry was used to study the intracellular levels of ROS in leukocytes (basally, as well as after in vitro stimulation with phorbol myristate acetate (PMA)), applying the probes dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) and the leukocyte expression of adhesion molecules CD62L and CD11b. ELISA was used to assess the plasma levels of soluble L-selectin, and TAS in plasma was measured using the ABTS+ reduction assay kit. RESULTS: The basal levels of ROS as well as PMA-stimulated ROS in leukocytes declined gradually during the ranger-training course, being lowest on the last day (P < 0.05). The level of TAS increased (P < 0.01) during the course. A striking decrease (P < 0.001) was observed in leukocyte CD62L expression and was sustained even after 3 d of recovery. The leukocyte expression of CD11b remained unchanged. CONCLUSION: The ranger-training course leads to a partial exhaustion of the leukocyte ROS-generating machinery and to a nearly total extinguishing of leukocyte CD62L expression. These changes may support the open-window hypothesis indicating reduced ability to combat microbial invasions before total restitution.     

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。