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1.
AIM:To investigate the inhibitory effect of thioredoxin 1 (Trx-1) over-expression on oxidative stress injury in 1-methyl-4-phenylpyridinium (MPP+)-induced rat pheochromocytoma PC12 cells by regulating NF-κB signaling pathway.METHODS:The PC12 cells were damaged by treatment with MPP+ at 1, 3 and 5 mmol/L, and the optimal concentration of 3 mmol/L was selected. The cell viability was measured by MTT assay. The oxidative stress indexes lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the cell culture supernatant were detected, and the protein expression of Trx-1 was determined by Western blot. Lentiviral infection with Ad-Trx-1-GFP sequence was used to establish a model of MPP+-treated PC12 cells with Trx-1 over-expression. The effects of Trx-1 over-expression on the cell viability, oxidative stress responses and NF-κB signaling pathway were determined by MTT assay, commercial kits and Western blot. The effects of phorbol 12-myristate 13-acetate (PMA), an activator of NF-κB signaling pathway, on the viability and oxidative stress of PC12 cells were observed. The NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) was used in MPP+-treated PC12 cells with Trx-1 over-expression, and the cell viability and oxidative stress responses were measured. RESULTS:The viability of PC12 cells, SOD activity in the supernatant and protein expression of Trx-1 were decreased, while LDH activity and MDA content in the supernatant were increased significantly by treatment with MPP+ at 1, 3 and 5 mmol/L. The effect of MPP+ at 3 mmol/L and 5 mmol/L was significantly greater than that at 1 mmol/L (P<0.05), and no significant difference between 3 mmol/L and 5 mmol/L was observed (P>0.05). The inhibitory effect of MPP+ on the viability of PC12 cells, and the oxidative stress injury and activation of NF-κB signaling pathway induced by MPP+ were significantly attenuated by over-expression of Trx-1. The inhibitory effect of MPP+ on the viability of PC12 cells and the oxidative stress injury induced by MPP+ were promoted by the activation of NF-κB signaling pathway, while the protective effects of Trx-1 over-expression on the MPP+-treated PC12 cells were enhanced by the inhibition of NF-κB signaling pathway. CONCLUSION:Over-expression of Trx-1 protects MPP+-treated PC12 cells from oxidative stress injury by regulating NF-κB signaling pathway.  相似文献   
2.
Trxf1是硫氧还蛋白家族中的一员,是组成硫氧还蛋白系统的重要组成部分,在植物的氧化逆境中发挥作用。为进一步研究该基因功能,通过RT-PCR方法从加工番茄叶片中克隆Trxf1基因的编码区,成功构建植物表达载体pBI121-Trxf1,采用农杆菌介导法转化番茄,经PCR、RT-PCR分析表明,植物表达载体已成功整合到番茄基因组中。实时荧光定量PCR研究表明,加工番茄Trxf1基因在其根、茎、叶、花等器官中均有表达,且在叶片中表达量最高。  相似文献   
3.
从二色补血草(Limonium bicolor)cDNA文库中分离出一个新的硫氧还蛋白过氧化物酶基因(LbTPx)全长cDNA序列.基因全长1016 bp,其中:5'非翻译区146 bp,3'非翻译区69 bp,开放读码框(ORF)801 bp,共编 码266个氨基酸;编码蛋白的分子质量为47.99× 10-24...  相似文献   
4.
AIM: To investigate the dynamic expression of thioredoxin 2 (Trx2) protein in the myocardial mitochondria under the condition of selenium deficiency. METHODS: A model of selenium deficiency was made using two-week-old SD rats. When the rats were fed for 20 weeks, 30 weeks and 40 weeks, the cardiac functions were detected by carotid artery intubation. The myocardial mitochondria were also extracted, and the protein expression of mitochondrial Trx2 was measured by Western blotting. The method of immunohistochemistry was used to detect Fas-associated death domain protein (FADD,a death marker protein) for determining the apoptosis of myocardial cells. The correlation between Trx2 and left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure rise(+dp/dtmax) and apoptotic index of the myocardial cells was analyzed. RESULTS: Compared with the corresponding period of normal control group, the expression of Trx2 protein in the selenium-deficiency rats was reduced. The expression of Trx2 protein was continuously reduced as the time of selenium deficiency prolonged. As the time of low-selenium feeding was going on, the systolic and diastolic functions of the heart were impaired, and the number of apoptotic myocardial cells was increased. The correlation analysis indicated that Trx2 had positive correlations with LVSP and +dp/dtmax, and had a negative correlation with the apoptotic index of myocardial cells. CONCLUSION: Selenium deficiency affects the expression of Trx2 protein,and causes impaired cardiac functions and the apoptosis of myocardial cells. Trx2 has a protective effect on myocardial cells.  相似文献   
5.
Macleaya cordata (plume poppy) is used in traditional Chinese medicine for its anti-inflammatory and antibacterial activities. In this study, we examined whether M. cordata extract and/or its major alkaloid constituents, protopine, allocryptopine, sanguinarine and chelerythrine activate the Nrf2 signalling pathway which regulates the expression of cytoprotective enzymes including heme oxygenase-1 (HO-1) and thioredoxin 1. In murine macrophage RAW264.7 cells, M. cordata extract increased both mRNA and protein levels of HO-1. Of the alkaloids examined, only sanguinarine appeared to be responsible for these effects. At the concentration of 2 μM, sanguinarine induced nuclear accumulation of Nrf2, increased the expression of Hmox1 gene encoding HO-1 and elevated HO-1 protein levels. Sanguinarine-induced Hmox1 mRNA expression was suppressed by SB203580, a pharmacologic inhibitor of p38 mitogen-activated protein kinases (p38 MAPKs). The upregulation of HO-1 in RAW264.7 cells by sanguinarine was, however, accompanied by decrease in cell viability. Nonetheless, sanguinarine at micromolar, non-cytotoxic concentrations elevated protein levels of HO-1 and thioredoxin 1 in primary cultures of human hepatocytes. We conclude that sanguinarine may, under appropriate conditions, increase the capacity of the enzymatic antioxidant defence system via activation of the p38 MAPK/Nrf2 pathway.  相似文献   
6.
反义trxs基因的导入对小麦种子发芽的影响   总被引:18,自引:0,他引:18  
刘雷  尹钧  任江萍  韩锦峰 《作物学报》2004,30(8):801-805
用基因枪将反义trxs基因导入小麦幼胚,经组织培养获得了抗性再生植株。PCR及限制酶切检测显示,反义trxs基因在转基因植株中基本完整。生化指标测定及发芽特性试验表明,与对照相比,转基因籽粒中Trx h活性明显降低;水溶蛋白和醇溶蛋白中巯基含量比率以及α-淀粉酶活性都有所下降;转基因种子的发芽势明显降低,但种子最终发  相似文献   
7.
紫杆柽柳cDNA文库构建与硫氧还蛋白基因的克隆   总被引:5,自引:0,他引:5  
以NaHCO3胁迫的紫杆柽柳(Tamarixandrossowii)为材料建立了cDNA文库。经检测,文库的初始滴度为5.7×105pfu,重组率为96%,插入片段的长度在0.3~3.0kb之间,平均长度为1.2kb,扩增后文库的滴度为4.0×109pfu/ml。对文库克隆进行随机测序,获得了紫杆柽柳的硫氧还蛋白(Thioredoxin)基因序列。生物信息学分析表明获得的硫氧还蛋白基因全长683bp,其中5'非翻译区长56bp,3'非翻译区长204bp,开放读码框长423bp,编码140个氨基酸,蛋白的分子量为15.1kD,理论等电点为5.59,其氨基酸序列中具有硫氧还蛋白的保守活性位点“WCGPC”序列,Clustal分析表明该硫氧还蛋白与蓖麻(Ricinuscommunis)的硫氧还蛋白氨基酸序列同源性高,达65%。本研究构建的cDNA文库为柽柳基因克隆和系统研究柽柳抗逆分子机理奠定了基础。  相似文献   
8.
为提高大麦种子中硫氧还蛋白h的活力,用基因枪法将硫氧还蛋白基因家族中与trxh基因高度同源的trxs基因导入4550块大麦幼胚愈伤组织,经过愈伤组织的诱导、筛选(PPT)和分化,得到49株T0代再生植株,并进行繁殖得到T1、T2代植株。采用PCR、嵌套PCR、酶切PCR产物和PCR-southern杂交多种方法对获得的植物材料进行检测。PCR检测显示,T0及其后代T1、T2代植株中都检测出了PCR阳性个体;嵌套PCR和酶切检测验证了T1、T2代植株中PCR产物与转化的目的片断相符。对转基因大麦籽粒进行了初步的生化分析,结果显示转基因大麦籽粒中硫氧还蛋白h的活性是非转基因籽粒的3.3倍;发芽后水溶蛋白和醇溶蛋白中巯基含量的比率都明显上升,其中水溶蛋白质中巯基含量比率是对照的1.5倍,醇溶蛋白质中巯基含量比率是对照的2倍。以上结果说明trxs基因在转基因大麦中能进行遗传并具有生物功能。  相似文献   
9.
AIM To investigate the protective effect of recombinant human serum albumin (HSA)-thioredox?in (Trx) fusion protein (HSA-Trx) on mice with acute lung injury (ALI) induced by influenza virus infection. METH?ODS: The recombinant HAS-Trx fusion protein was generated by Pichia pastoris expression system. ICR mice were used to establish the animal model of ALI induced by PR8 (H1N1) influenza virus, and the experimental mice were divided into healthy control group, ALI group, ALI+Trx group and ALI+HSA-Trx group, with 10 mice in each group. The bronchoalveolar lavage fluid (BALF) in each group was collected, the total number of cells and the number of alveolar neutrophils were determined, the protein concentration was measured by Coomassie brilliant blue solution method, and the interferon-γ (IFN-γ) content in BALF was detected by ELISA. The lung tissues were collected for hematoxylin and eosin staining. The inducible nitric oxide synthase (iNOS), 8-hydroxydeoxyguanosine (8-OHdG) and 3-nitrotyrosine (NO2-Tyr) in lung tissues were detected by immunofluorescence method. Peroxide concentration in plasma was evaluated using a CR2000RC analyzer. RESULTS HSA-Trx treatment significantly reduced the total number of cells, neutrophils and total protein in BALF of ALI mice (P<0.05), and decreased the levels of 8-OHdG, NO2-Tyr in lung tissue and peroxide in plasma (P<0.05). However, it has no significant inhibitory effect on iNOS and IFN-γ expression (P>0.05). CONCLUSION HSA-Trx inhibits inflammatory response and excessive production of nitric oxide in the lung, thus protecting influ?enza virus-induced ALI mice.  相似文献   
10.
本研究以绵羊睾丸组织为材料,利用RT-PCR技术获得绵羊硫氧还蛋白类蛋白2(Thioredoxin like protein 2,Txl-2)基因的CDS区序列,并对其进行生物信息学分析。结果表明,克隆得到的绵羊Txl-2基因CDS区为795 bp,编码264个氨基酸。生物信息学分析结果表明,Txl-2编码的蛋白无跨膜区、信号肽和N-糖基化位点,存在多个磷酸化位点;预测出了Txl-2蛋白的二级结构和三级结构;Clustel W方法对比绵羊Txl-2与绵羊、山羊预测序列同源性最高。  相似文献   
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