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1.
人粒-巨噬细胞集落刺激因子(hGM-CSF)是一种重要的造血生长因子.利用基因重组技术构建两个hGM-CSF的E.coli表达菌株,一个为在不改变氨基酸顺序的前提下,对mRNA翻译起始区核苷酸顺序进行优化突变(hGM-CSF(M)),另一个为未突变的对照(hGM-CSF(N)).经酶切电泳、DNA测序、SDS-PAGE和Westernblot等分析鉴定,证明两者均能表达特异性的14.6kDhGM-CSF,但hGM-CSF(M)的表达水平较hGM-CSF(N)提高了1.26倍,占菌体总蛋白的16.9%.mRNA翻译起始区二级结构预测分析表明,优化突变后生成自由能ΔG从原来的-10.2提高至-9.4Kcal,AUG从部分配对状态变为非配对状态.  相似文献   

2.
白细胞介素(IL-1)及阿片肽作为神经调质参与了神经细胞兴奋性毒性作用,以大鼠大脑皮层神经细胞为研究对象,探讨了IL-1、阿片肽和c-fos、c-jun表达产物之间的关系,结果表明,IL-1β能诱导大脑皮层神经细胞c-fos、c-jun mRNA瞬时短暂表达,15min增高,30min达高峰,c-fos mRNA 2h回至基线水平,c-jun m RNA 8h回至基线水平;联合应用c-fos,c-  相似文献   

3.
人粒-巨噬细胞集落刺激因子(hGM-CSF)是一种重要的造血生长因子。利用基因重组技术构建两个hGM-CSF的E.coli表达菌株,一个为在不改变氨基酸顺序的前提下,对mRNA翻译起始区核苷酸顺序进行优化突变(hGM-CSF(M)),另一个为未突变的对照(hGM-CSF(N))。经酶切电泳、DNA测序、SDS-PAGE和Western blot等未突变的对照(hGM-CSF(N))。经酶切电泳、D  相似文献   

4.
通过培养的人主动脉平滑肌细胞(hASMC)及脐静脉内皮细胞(hUVEC),应用3H-TdR参入、Northernblot分析、逆转录多聚酶链反应(RT-PCR)、放射免疫分析(RIA)、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖(HSPG)对hASMC和hUVECDNA合成的作用及对血小板源生长因子(PDGF)、PDGF受体、转化生长因子β(TGF-β)、内皮素-1(ET-1)或碱性成纤维细胞生长因子(bFGF)基因表达和肾素-血管紧张系统(RAS)的影响,结果显示,HSPG明显抑制培养的hASMC基础的DNA合成(cpm值为:10385±3263vs,25541±6421,P<0.01)及外源性PDGF诱导的DNA合成(cpm值为:9878±1947vs.13481±44l0,P<0.05);抑制PDGFA链、TGF-Bp和ET-1mRNA表达,提高PDGFa和β受体mRNA的表达;显著降低hASMC培养液中血管紧张素Ⅱ(AngⅡ)的浓度和血管紧张素转换酶(ACE)的活性,推测HSPG抑制PDGFA链、TGF-β及ET-1mRNA表达,降低ACE活性及AngⅡ浓度是其抑制hASMC增殖的重要机  相似文献   

5.
高氧预适应对大鼠心肌缺血损伤时抗氧化酶的影响   总被引:1,自引:0,他引:1  
抗氧化酶具有减轻心肌缺血再灌注损伤的作用,在抗氧化酶中,比较重要的是超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GlutathionePeroxidase,GSHpx)和过氧化氢酶(CAT)。为了解高氧预适应(HyperoxicPreconditioning,HOP)对大鼠心肌缺血损伤时抗氧化酶的影响,本实验将实验组大鼠放入高压氧舱内,每日吸80-85%氧气(1atm,15-20%为氮气)6h,连续7d。利用Langendorf装置做成心肌缺血再灌注模型。实验动物随机分为二个部分。第一部分可逆性心肌缺血(HOPA组与对照A组):缺血10min,再灌注60min。观察冠脉回流液中SOD活力,检测心肌内抗氧化酶活力(SOD,GSHpx,CAT)。第二部分不可逆性心肌缺血(HOPB组与对照B组):缺血60min,再灌注60min。测定冠脉回流液中肌酸磷酸激酶(CPK)含量,SOD及心肌内抗氧化酶活力。结果表明:对于可逆性心肌缺血:SOD,GSHpx活力升高;对于不可逆性心肌缺血损伤:HOP能减少CPK释放,SOD活力升高。  相似文献   

6.
大鼠液压冲击脑损伤脑干c-fos mRNA表达的定位观察   总被引:2,自引:0,他引:2  
目的:研究大鼠中度侧位液压冲击脑损伤时脑干c-fos mRNA及其表达产物Fos变化规律。方法:雄性SD大鼠,随机分为正常对照组、手术对照组和损伤组。损伤组动物均给以0.2MPa液压冲击脑损伤,按冲击后处死时间不同再分为5min、15min、30min、1h、2h、4h、8h和12h组。应用免疫组织化学和原位杂交方法观察c-fos在脑干的表达。结果:脑冲击后15min-12h,Fos阳性细胞数逐渐  相似文献   

7.
重组人GM—CSF基因在昆虫细胞中的表达   总被引:1,自引:0,他引:1  
利用苜蓿夜蛾核型多角体病毒(AcNPV)带β-Galactosidase基因标记的非融合蛋白基因转移载体pBlueBac将人粒细胞巨噬细胞集落刺激因子(hGM-CSF)基因成功地插入病毒AcNPV的基因组中.hGM-CSF基因在感染重组病毒的草地夜蛾(Spodopterafrugiperda)培养细胞Sf9中得到表达,感染后的Sf9细胞培养液能刺激人骨髓细胞在体外形成典型的集落,表达水平可达2.7×1055CFU/ml。以hGM-CSF单抗所作的WesternBlotting表明,表达的hGM-CSF对是3种糖基化程度不同的产物,分子量分别约为15kd,18kd和20kd。  相似文献   

8.
为了研究甲状旁腺素(parathyroidhormone,PTH)促成骨样细胞ROS17/2.8增殖分化的信号传递的机理,观察了PTH对细胞内酪氨酸蛋白激酶(tyrosineproteinkinases,TPK)活性及酪氨酸蛋白磷酸化水平的影响,并检测了PTH对c-rasmRNA表达的诱导作用.结果表明,PTH可增强细胞内TPK活性,提高细胞内酪氨酸蛋白磷酸化水平,以及促进c-rasmRNA的持续表达.这些结果提示,PTH促增殖的细胞内信息传递通路与酪氨酸蛋白激酶-ras-MAPKinase通路密切相关,而PKA/PKC通路的最终效应可能是通过与上述通路的crosstalk实现的  相似文献   

9.
洞庭湖滨湖湿地平原农村生态工程初步研究严斧,游有林,刘劲凡,卢源德,周为民(中国科学院生态环境研究中心系统生态室沅江课题组,大庸市416600)RuralEcologicalEngineeringonWetlandPlainofDongtingLakeside¥.YanFu;YouYoulin;LiuJinfan;LuYuande;ZhouWeimin(YuanjiangResearchGroup,ResearchCenterforEco-environmentalScience,A-cademiaSinica,Dayong416600).ChineseJournalofEcology,1993,12(2):45—48.AccordingtothemaintypesofresourcecombinationsonwetlandplainofDongtinglakeside,severaleco-logicalengineeringsincludinglakesidereed-forest,lowlandrice-fish-ramie,rnanure-forage-fuel,fish-pig-duckandf  相似文献   

10.
用Kunkel突变法,将单链尿激酶型纤溶酶原激活剂(scu-PA)cDNA基因中编码Pro155—Lys158的片段定点突变,并将此突变的scu-PA(tscu-PA)的cDNA克隆到表达载体pCM-β-neo中,与pCM-dhfr共转染CHO/DHFR-细胞.获得的稳定表达株在无血清培养基中24h的表达量为620IU/106细胞.经锌离子螯合Sepharose亲和层析得到tscu-PA纯品.SDS-PAGE显示tscu-PA分子量为53kD左右,与预期的结果相符.tscu-PA是由凝血酶激活而不是由纤溶酶激活,但激活后也能转变为双链分子(tcu-PA).tscu-PA仍保持了scu-PA的血纤维蛋白亲和性.酶动力学研究表明,激活后的tscu-PA水解S2444的Km和Kcat值与高分子量尿激酶(HUK)相似.体外溶栓实验结果表明,tscu-PA可以选择性地溶解富含凝血酶的血凝块,对贫凝血酶的血凝块作用不大  相似文献   

11.
黄芩黄酮对硒性白内障晶状体抗氧化酶表达的影响   总被引:9,自引:0,他引:9  
为探讨黄芩黄酮防治白内障的作用机理 ,采用半定量RT PCR方法比较正常组、白内障组和中药防治组大鼠晶状体中GSH Px、GR和Cu ZnSOD的mRNA水平 .白内障组GSH Px、GR和Cu ZnSOD的mRNA水平在 15d龄时显著高于正常 ,然后下降 ;在 2 7d和 31d龄 ,GR和Cu ZnSOD的mRNA水平下降至与正常无显著差异 ,GSH PxmRNA水平仍略高于正常 .中药防治组晶状体中 ,3种抗氧化酶的mRNA水平在各实验取样点无明显变化 ;其中 ,GR和Cu ZnSOD的mRNA水平一直与正常无显著差异 ,GSH PxmRNA水平略高于正常 .黄芩黄酮可能通过有效清除亚硒酸钠间接产生的活性氧来防止白内障的发生 ,并使亚硒酸钠对晶状体抗氧化酶表达的影响得以消除  相似文献   

12.
The effect of (1-benzoyl-1H-indazol-3-yl)oxylacetate L-Lysine (bendazac-lysine) on some enzymatic activities involved in the metabolism of reduced glutathione (GSH) was studied in the rabbit lens during developing cataract induced by a single dose of X-rays (2000 rads). The specific activities of glutathione reductase (G.R.), glutathione peroxidase (GSH.Px) and glutathione S-transferase (GSHS-tr.) do not change following irradiation and treatment with bendazac-lysine. The activity of the same enzymes expressed as a function of water soluble proteins (WSP) per lens significantly decreases (P less than 0.01) as compared to controls in the irradiated lens not treated with bendazac-lysine (ILNTB) at the 8th week, whereas no significant decrease as compared to controls is observed in the irradiated lens treated with bendazac-lysine (ILTB). In the ILNTB the specific activity of glucose-6-phosphate dehydrogenase (G6PDH) is reduced by 10% after 0.3 weeks and by 29% after 12 weeks. In the ILTB the specific activity of G6PDH is reduced by 8% after 0.3 weeks and by 14.5% after 12 weeks. The specific activity of superoxide dismutase (SOD) in the ILNTB is reduced by 19% after 0.3 weeks and reached 31% after 12 weeks. In the ILTB the specific activity of SOD is reduced by 11% after 0.3 weeks and 19.8% after 12 weeks. The mechanism of protective effect of bendazac-lysine on cataract is discussed.  相似文献   

13.
Expression of antioxidant enzymes in human inflammatory cells   总被引:5,自引:0,他引:5  
Because antioxidant enzymesmay have an important role in the oxidant resistance ofinflammatory cells, we investigated the mRNA levels and specificactivities of manganese and copper-zinc superoxide dismutases (Mn SODand Cu,Zn SOD), catalase (Cat), and glutathione peroxidase, as well asthe concentrations of glutathione (GSH) in human neutrophils,monocytes, monocyte-derived macrophages, and alveolar macrophages.Levels of GSH and glutathione peroxidase activity in monocytes werethree times higher than in neutrophils, whereas the mRNA of Cat was50-fold and its specific activity 4-fold higher in neutrophils.Although Mn SOD mRNA levels were higher in neutrophils, enzymeactivities, as well as those of Cu,Zn SOD, were similar in allphagocytic cells. Neutrophils lost their viability, assessed by adeninenucleotide depletion, within 24 h ex vivo and more rapidly if GSH wasdepleted. However, neutrophils were the most resistant cell type toexogenousH2O2.In conclusion, high Cat activity of neutrophils appears to explaintheir high resistance against exogenousH2O2,whereas low GSH content and GSH-related enzymes seem to account for thepoor survival of human neutrophils.

  相似文献   

14.
Thirty-two barrows (Duroc x Landrace x Yorkshire) were randomly divided into four groups, each of which included eight pigs. The groups received the same basal diet supplemented with 0, 100, 250 and 400mg/kg fluoride, respectively. The malondialdehyde (MDA) and glutathione (GSH) levels, antioxidant enzymes activities and zinc/copper superoxide dismutase (Cu/Zn SOD) mRNA content in the liver were determined to evaluate the fluoride hepatic intoxication. Results showed the increased lipid peroxides (LPO) level and the reduced GSH content, along with a concomitant decrease in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px). Moreover, the level of hepatic Cu/Zn SOD mRNA was also significantly reduced. We suggest the mechanism of fluoride injuring the liver as follows: fluoride causes a decrease in Cu/Zn SOD mRNA and the reduced activities of antioxidant enzymes, leads to the declined ability of scavenging free radicals with excessive production of LPO, which seriously damages the hepatic structure and function.  相似文献   

15.
In the present study, we have assayed the enzymatic activity of Cu,Zn–SOD, Mn–SOD, GSH–Px, GSH-Red, Cat, and G6PD in rat retina as a function of age. Conjugated diene levels and MDA formation were also determined. The conjugated diene levels in rat retina were found to increase significantly with age, accompanied by a marked decrease in GSH–Px and Cat activities. No agerelated change in MDA levels and in GSH-Red and G6PD activity was found, whereas a significant increase in SOD activity was observed between 1 and 4 months. Decreased GSH–Px and Cat activity is related to increased lipid peroxidation with age.  相似文献   

16.
萝卜硫素(sulforaphane,SFN)是一种在十字花科植物中含量丰富,且具有抗氧化效应的天然物质。本文基于核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)介导的抗氧化系统,探究不同时长低温暴露对骨骼肌抗氧化酶的影响及SFN对低温暴露骨骼肌抗氧化能力的作用。首先,30只雄性C57BL/6N小鼠随机分为常温对照组(0 h组)、低温暴露1 h组(1 h组)和低温暴露3 h组(3 h组)。其次,40只雄性C57BL/6N小鼠随机分为PBS常温对照组(PBS+Con),PBS低温暴露3 h组(PBS+Cold),SFN常温对照组(SFN+Con)和SFN低温暴露3 h组(SFN+Cold)。小鼠在急性温度干预前腹腔注射4次SFN或等体积PBS。急性低温暴露后,取小鼠骨骼肌,试剂盒检测活性氧(ROS)水平、总抗氧化能力(T-AOC)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量;荧光实时定量PCR检测Nrf2介导的抗氧化酶和参与生成谷胱甘肽相关酶的mRNA转录水平;Western blot检测Nrf2介导的抗氧化酶蛋白表达。结果显示,与0和1 h组相比,3 h组小鼠骨骼肌Nrf 2和抗氧化酶基因(Gpx 1、Hmox1、Cat、Sod 1和Nqo 1)的mRNA转录水平显著降低,ROS水平显著增加。与PBS+Con组相比,PBS+Cold组小鼠骨骼肌Nrf2和抗氧化酶(HMOX1和CAT)蛋白表达、GSH/GSSG比值及T-AOC水平显著降低,而GSSG含量和ROS水平增加。与PBS+Cold组相比,SFN+Cold组小鼠骨骼肌Nrf 2 mRNA及其蛋白表达、抗氧化酶(HMOX1和SOD1)蛋白表达、抗氧化酶基因(Gpx 1、Hmox 1、Cat、Sod 1和Nqo 1)mRNA转录水平、参与GSH生成的酶基因(Gclm和Gss)mRNA转录水平、GSH/GSSG比值以及T-AOC水平显著提高,而GSSG含量和ROS水平显著降低。综上,3 h急性低温暴露降低了Nrf2介导的抗氧化作用。而低温暴露前给予SFN补充,则激活了Nrf2介导的抗氧化酶和谷胱甘肽抗氧化系统,增强了骨骼肌抗氧化能力。  相似文献   

17.
The aim of this study was to examine changes in activities of cytochrome c oxidase (CCO), glucose-6-phosphate dehydrogenase (G6PDH), Cu–Zn superoxide dismutase (Cu–Zn SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) levels and copper (Cu), zinc (Zn) and selenium (Se) concentrations, and to assess the possible differences between preterm placentas, placentas from term pregnancies complicated by intrauterine growth restriction (IUGR) and full-term control placentas.The enzyme activities and the level of GSH decreased in IUGR and preterm placentas in comparison with the control group. CCO activity and GSH level in preterm placentas were markedly lower compared with the IUGR (P<0.01; P<0.05) and control (P<0.01; P<0.05) placentas, respectively. In IUGR placentas the level of Cu was reduced by 23% (P<0.05) and Zn by 37%. In preterm placentas the level of Cu was reduced by 19% and Zn by 42%. Se level in IUGR and preterm placentas was higher (P<0.05) by 28% and 32% than in control group, respectively. The strong relation was observed between birth weight and CCO activity, birth weight and Cu–Zn SOD activity, and a low level of Zn and Cu influenced the birth weight especially in IUGR cases. Moreover, the strong inverse correlation between Se level and birth weight, Se level and placental weight and Se level and CCO activity are new findings.  相似文献   

18.
The expression patterns of different genes encoding antioxidant enzymes and heat shock proteins were investigated, in present study, by real-time quantitative PCR in the hepatopancreas of abalone Haliotis discus hannai fed with different levels of dietary zinc (6.69, 33.8, 710.6 and 3462.5 mg/kg) for 20 weeks. The antioxidant enzymes include Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT), mu-glutathione-s-transferase (mu-GST) and thioredoxin peroxidase (TPx). The results showed that the mRNA expression of these antioxidant enzymes increased and reached the maximum at the dietary zinc level of 33.8 mg/kg, and then dropped progressively. Expression levels of the heat shock proteins (HSP26, HSP70 and HSP90) firstly increased at 33.8 mg/kg dietary Zn level, and reached to the maximum at 710.6 mg/kg, then dropped at 3462.5 mg/kg (p<0.05). Excessive dietary Zn (710.6 and 3462.5 mg/kg) significantly increases the Zn content and significantly decreases the total antioxidant capacity (T-AOC) in hepatopancreas (p<0.05). These findings showed that dietary Zn (33.8 mg/kg) could highly trigger the expression levels of antioxidant enzymes and heat shock proteins, but excessive dietary Zn (710.6 and 3462.5 mg/kg) induces a high oxidative stress in abalone.  相似文献   

19.
Abstract: Nerve growth factor (NGF) is a member of the neuro- trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per- oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.  相似文献   

20.
Ninety-six castrated boars (Duroc x Landrace x Yorkshire) were randomly divided into four groups, each of which was replicated three times with eight pigs. The groups received the same basal diet supplemented with 0, 5, 10, and 20 mg/kg lead, respectively. The malondialdehyde and glutathione levels, antioxidant enzymes activities, and zinc/copper superoxide dismutase (Zn/Cu SOD) mRNA content in the liver were determined to evaluate the lead hepatic intoxication caused by the lead. Results showed the increased lipid peroxides level and the reduced glutathione content, along with a concomitant decrease in the activities of superoxide dismutase, catalase, and glutathione peroxidase. Moreover, the level of hepatic Zn/Cu SOD mRNA was also significantly reduced. We suggest potential mechanism for lead intoxication in liver as follows: lead causes parallel decrease in Zn/Cu SOD mRNA and activities of antioxidant enzymes, leading to the declined ability of scavenging free radicals with excessive production of lipid peroxides, which seriously damages the hepatic structure and function.  相似文献   

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