硒对动物基因表达的调控

硒是动物机体必需的微量元素,其通过多种方式参与调解动物基因的表达。本文简述了硒在动物体内的调节机制,并从硒对谷胱甘肽过氧化物酶、碘甲状腺素5’脱碘酶(ID I)以及其他基因表达的调控,硒的抗癌作用,硒对硒磷酸的调控等几方面阐述了硒在动物基因表达调控方面的研究现状。     

饲粮硒来源及添加水平对仔猪组织中细胞内谷胱甘肽过氧化物酶基因mRNA表达的影响

本文旨在研究饲粮中硒来源及添加水平对仔猪细胞内谷胱甘肽过氧化物酶(GPX1)mRNA表达的影响,为合理利用各种硒源提供一定的理论基础.选择35日龄体重相近的三江白猪仔猪90头,随机分为10组,每组3个重复,每个重复3头猪.分别在9个试验组饲粮中添加纳米硒、酵母硒、亚硒酸钠,其剂量以硒计,添加量分别为0.1、0.3、0.5 mg/kg 3个水平,对照组饲粮中不另添加硒.试验期为35 d,试验结束后采用反转录RT-PCR技术测定仔猪肝脏、肾脏、脾脏、肌肉组织中GPX1 mRNA的表达量.结果表明:1)在肝脏组织中,0.5 mg/kg纳米硒组GPX1 mRNA表达量显著高于亚硒酸钠组(P<0.05),0.5 mg/kg纳米硒组和0.3 mg/kg酵母硒组显著高于对照组(P<0.05);2)在肾脏组织中,各试验组之间GPX1 mRNA表达量差异均不显著(P>0.05);3)在脾脏组织中,0.5 mg/kg纳米硒组GPX1 mRNA表达量显著高于酵母硒组和亚硒酸钠组(P<0.05),0.5 mg/kg纳米硒组和0.3 mg/kg酵母硒组显著高于对照组(P<0.05);4)在肌肉组织中,0.5 mg/kg纳米硒组GPX1 mRNA表达量显著低于亚硒酸钠组和对照组(P<0.05).由此可见,在饲粮中添加0.5 mg/kg纳米硒能够提高仔猪肝脏、脾脏中GPX1 mRNA的表达量,而肌肉组织中的表达水平则低于亚硒酸钠组和对照组.     

富硒益生菌对小鼠肝脏硒含量、GPX活性和GPX-1mRNA表达的影响

为了研究富硒益生菌对小鼠肝脏硒含量、谷胱甘肽过氧化物酶(GPX)活性和GPX-1 mRNA表达量的影响,试验将60只昆明系小鼠(雌雄各半)随机分为对照组、亚硒酸钠组和富硒益生菌组,每天分别灌胃纯化水、亚硒酸钠和富硒益生菌、益生菌各0.5 m L,连续28 d,试验结束时测定肝脏硒含量、GPX活性和GPX-1 mRNA表达量。结果表明:富硒益生菌组和亚硒酸钠组小鼠肝脏硒含量和GPX活性均极显著高于对照组(P0.01),而富硒益生菌组小鼠肝脏硒含量和GPX活性显著高于亚硒酸钠组(P0.05);富硒益生菌组和亚硒酸钠组小鼠肝脏GPX-1 mRNA的表达水平与对照组相比分别高229%、216%,而富硒益生菌组小鼠肝脏GPX-1 mRNA的表达水平与亚硒酸钠组相比高4.11%。     

硒和硒蛋白调节动物抗氧化功能及硒在畜牧生产中应用的研究进展

谷胱甘肽过氧化物酶1(Glutathione Peroxidase1,GPX1)、硫氧还蛋白还原酶(Thioredoxin Reductase,TrxR)/硫氧还蛋白(Thioredoxin,Trx)是在机体中发挥抗氧化功能的硒蛋白。GPX1可提高动物抗氧化能力,抑制核因子кB(Nuclear Factor Kappa B,NF-кB)信号通路,减缓细胞氧化损伤,激活核因子E2相关因子2/抗氧化反应元件通路,提高Trx和TrxR蛋白活性,增强Trx/TrxR系统抗氧化功能,降低促炎因子和活性氧等产生,维持机体氧化还原稳态。因此,本文着重阐述GPX1、Trx与TrxR对动物抗氧化系统的调控作用及硒在畜牧生产中的应用进展,为硒及硒蛋白增强动物抗氧化功能方面的研究及硒在畜牧生产中的应用提供理论参考。     

日粮添加硒与维生素E对公山羊组织中GPX基因mRNA表达量的影响

为了探讨硒与维生素E含量与山羊谷胱甘肽过氧化物酶(GPX)基因表达的关系,试验选择60日龄、体重(15.21±2.62)kg的山羊公羔羊56只,分成7组(G0～G6组),每组8只,G0为对照组,饲喂基础日粮,G1～G6为试验组,分别饲喂3个硒水平(0.2 mg/kg、0.3 mg/kg、0.4 mg/kg)和2个维生素E水平(100 IU/kg、200 IU/kg)的组合日粮,试验期为120 d,试验结束当天每组选取4只公山羊剖杀后取肝脏、心脏、脾脏、肺脏、睾丸、肾脏,分析GPX基因(GPX-1、GPX-2、GPX-3和GPX-4)mRNA的表达量。结果表明:添加硒和维生素E后,同对照组相比,硒和维生素E的加入促进了GPX基因的表达;当日粮中硒添加量为0.3 mg/kg、维生素E添加量为100 IU/kg时,GPX-1、GPX-2、GPX-3和GPX-4基因mRNA在各组织中的表达量最高。说明添加硒和维生素E可促进GPX基因的表达,且硒添加量为0.3 mg/kg、维生素E添加量为100 IU/kg时效果较好。     

肉鸡饲料中的硒来源

加拿大圭尔夫大学Steve Leeson教授等进行试验比较了3种Se来源（亚硒酸钠，优质硒酵母和B—TRAXIM Se）对肉种鸡和产蛋母鸡的生物效力。饲喂亚硒酸钠或硒酵母肉种鸡的肝谷胱甘肽过氧化物酶（GPX）的活性更高（P〈0．01），     

牦牛GPX5基因CDS区的克隆和表达研究

GPX5作为谷胱甘肽过氧化物酶的成员之一,表达于牦牛附睾之中,其主要作用为抗氧化应激。本实验旨在通过分子技术对牦牛GPX5基因进行克隆,分析其生物信息学的特征和附睾组织的表达特点。结果显示:牦牛GPX5基因CDS区有630 bp、209个氨基酸被编码,所编码蛋白带正电,具有亲水性且不稳定,无跨膜域却存在信号肽;GPX5基因在附睾头的表达高于附睾的颈和尾(P<0.01)。牦牛附睾中的GPX5能有效抵抗氧化应激以保护精子。     

硒与大鼠甲状腺激素代谢的关系及其对心肌肌球蛋白重链基因表达的影响

应用心肌肌球蛋白重链（ＣＭＨＣ）基因探针分析了克山病病区粮（硒缺乏）和病区粮补硒喂养大鼠心肌中α和β－ＣＭＨＣ基因表达的变化，并观测了大鼠心肌Ｔ４５′－脱单碘酶活性和Ｔ３水平的改变。结果表明，日粮缺硒可导致大鼠ＣＭＨＣ基因表达的偏移，心肌Ｔ４５′－脱单碘酶活性下降和心肌Ｔ３水平的降低。病区粮补加硒后，上述改变明显向正常方向逆转。给病区粮喂养大鼠腹腔注射外源性Ｔ３，可使α－ＣＭＨＣｍＲＮＡ水平明显升高，而β－ＣＭＨＣｍＲＮＡ水平下降，表明Ｔ３对大鼠ＣＭＨＣ基因的表达具有一定的调控作用，提示硒可通过Ｔ４５′－脱单碘酶→Ｔ３途径对ＣＭＨＣ基因表达进行调节     

牦牛GPX基因家族生物信息学及表达分析

本研究旨在筛选F1代雄性不育犏牛与正常牦牛谷胱甘肽过氧化酶家族（GPXs）差异表达基因（DEGs）,探索在牦牛附睾精子成熟过程中可能发挥重要作用的调控基因和生物学功能。从转录组测序数据库中筛选并鉴定得到牦牛GPXs基因,利用生物信息学方法分析GPXs基因理化性质、基因结构、系统进化关系、表达情况以及GPXs蛋白多序列比对、二级结构、三级结构。结果显示：共鉴定了8个牦牛GPXs基因（GPX1～GPX8）,分子量为22.60～56.31 ku,等电点为5.09～5.32,包含4～8个保守元件;GPXs蛋白具有共同保守氨基酸序列,在蛋白二、三级结构分析中显示都存在反向平行的β-折叠结构,并与ALOX5、GSS、GSTM3蛋白均存在相互作用;系统进化树显示,GPX基因家族在反刍动物的同源性最高;牦牛和犏牛附睾组织检测到GPX2、GPX3、GPX5、GPX8基因表达存在显著性差异。研究表明,牦牛GPXs基因家族结构高度保守,功能上可能参与调控牦牛附睾精子抗氧化等过程。     

GPX4失活通过JNK通路缓解LPS诱导巨噬细胞炎症反应

【目的】 研究硒蛋白谷胱甘肽过氧化物酶4（glutathione peroxidases 4,GPX4）失活如何参与调控脂多糖（lipopolysaccharide,LPS）诱导的RAW264.7巨噬细胞炎症反应及其潜在的分子机制。【方法】 体外培养RAW264.7巨噬细胞,以DMSO为对照,使用0.1~5.0 μmol/L GPX4抑制剂FIN56处理,通过CCK-8法检测细胞活力和Western blotting检测GPX4蛋白表达水平,确定抑制剂最适浓度。将RAW264.7巨噬细胞分为4组：对照组,添加DMSO培养24 h;FIN56（GPX4抑制剂）组,添加0.5 μmol/L FIN56培养24 h;DMSO-LPS组,DMSO培养24 h后使用LPS （100 ng/mL）刺激3 h;FIN56-LPS组,FIN56培养24 h后使用LPS刺激3 h。各组细胞经培养后,利用荧光探针2',7'-二氯二氢荧光素二乙酸酯（2',7'-dichlorodi-hydrofluorescein diacetate,H₂DCFDA）检测细胞内活性氧（reactive oxygen species,ROS）水平,使用试剂盒法检测细胞内丙二醛（malondialdehyde,MDA）水平,分别使用ELISA和荧光定量PCR检测促炎细胞因子白细胞介素1β（interleukin-1β,IL-1β）、白细胞介素6（interleukin-6,IL-6）、肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）的分泌水平和基因表达量,使用Western blotting法检测Toll样受体4（toll like receptor 4,TLR4）信号通路相关蛋白表达水平。【结果】 与DMSO处理相比,0.5 μmol/L FIN56处理巨噬细胞24 h对细胞活性无显著影响（P>0.05）,且显著降低GPX4蛋白表达水平（P<0.05）,故选用0.5 μmol/L FIN56用于后续实验。与对照组相比,FIN56和LPS均显著增加了ROS积累（P<0.05）,而与DMSO-LPS组相比,FIN56-LPS组ROS水平显著升高（P<0.05）。与对照组相比,LPS可显著增加小鼠巨噬细胞IL-1β、IL-6和TNF-α的mRNA表达量和IL-6含量,以及c-Jun氨基末端蛋白激酶（c-Jun N-terminal protein kinase,JNK）和c-Jun磷酸化水平（P<0.05）,而FIN56可显著下调LPS诱导的IL-6的mRNA表达量和含量及JNK和c-Jun磷酸化水平（P<0.05）,但对p38蛋白（p38 MAPK,p38）、细胞外调节蛋白激酶（phosphorylate extracellular regulated protein kinases1/2,Erk1/2）蛋白表达水平无显著影响（P>0.05）。【结论】 GPX4失活可有效阻断JNK和c-Jun磷酸化,从而特异性抑制LPS诱导的巨噬细胞的炎症反应。该结果可为以GPX4为靶点研发抗炎治疗策略提供理论依据。     

猪GPX5,FUT1和PRLR基因的遗传多态性及遗传效应分析

为了分析谷胱甘肽过氧化物酶5基因(Glutathion Peroxidase 5,GPX5),岩藻糖转移酶1基因(α1-Fucosytransferase,FUT1)和催乳素受体基因(Prolactin receptor,PRLR)在野猪与大白猪杂交的F1代群体中的遗传多态性及遗传效应。采用PCR-RFLP方法检测了GPX5,FUT1和PRLR基因的多态性,分析其对初生重(LiB)和30日龄体重(Li30)的遗传效应。结果表明,除了PRLR基因外,其余2个基因的基因频率与大白猪已有研究结果基本一致;PRLR基因的AA型个体在Li30上比AB和BB型个体可显著增重1.42～1.47 kg(P0.05);GPX5和FUT1基因对这2个性状的效应均不显著。结果提示,野猪血统的引入没有显著改变现有猪群的遗传基础,PRLR基因可在生产中考虑应用。     

Effects of selenium supplementation on the oxidative state of acute heat stress‐exposed quails

This study aimed to evaluate the effect of heat stress (HS) and selenium supplementation on markers of stress, meat quality and gene expression. For this, meat quails of 42 days of age were fed a diet that either met [0.33 mg/kg, nutritional demand for selenium (SS)] or did not meet [0.11 mg/kg, selenium deficient (SD)] the nutritional demands for selenium during the 7 days of evaluation. In addition, the animals were kept at either a thermal comfort temperature (25 °C) or exposed to HS (38 °C for 24 h). Glutathione synthetase (GSS), glutathione reductase (GSR) and uncoupling protein (UCP) gene expression were influenced by the interaction between temperature and diet. Animals subjected to HS and fed the SS diet exhibited the highest GSS and GSR gene expression. In terms of UCP gene expression, the lowest values were observed in HS animals on the SD diet. Glutathione peroxidase 7 (GPX7) gene expression, body temperature (BT) and creatine kinase (CK) activity were influenced by both selenium supplementation and HS. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and creatinine content all were influenced by the diet/environment interaction. The highest AST activity, ALT activity and creatinine levels were observed in animals that were both on the SD diet and exposed to HS. HS animals also exhibited an increased heterophil/lymphocyte ratio and lower triiodothyronine (T3) hormone levels than birds that remained at the comfortable temperature. Animals subjected to HS and fed with selenium supplemented diet showed better results regarding gene expression and, thus, better results for the activities of enzymes used as stress markers, which could be due to the higher antioxidant capacity provided by the action of the studied genes.     

葡萄糖对鹅原代肝细胞GPX1和SOD1基因表达及酶活力的影响

In order to investigate the effect of glucose on oxidative stress in cultured goose primary hepatocytes, partial gene sequences of GPX1 and SOD1 genes were cloned, and the goose primary hepatocytes were treated with different concentrations（0（control group）, 5, 25 and 35 mmol/L） of glucose for 48 h, and then enzyme activities of GPX and SOD and mRNA levels of GPX1 and SOD1 were examined. The results showed that goose GPX1 gene sequence had the highly similarity with Zebra Finch, and SOD1 had the highly similarity with Red Jungle fowl. Compared with the control group, 5 mmol/L glucose had no significant effect on GPX enzyme activity （P>0.05）, but 25 and 35 mmol/L glucose could significantly decrease GPX enzyme activity (P＜0.05).5 mmol/L glucose could significantly increase SOD enzyme activity （P＜0.05）, but 25 and 35 mmol/L glucose had no significant effects on SOD enzyme activity（P＞0.05）.35 mmol/L glucose could significantly increase mRNA expression of GPX1 and SOD1 genes （P＜0.05）, but 5 and 25 mmol/L glucose had no significant effects on the two genes（P＞0.05）.In conclusion, the high concentration glucose （35 mmol/L） could induce intracellular oxidative stress in goose primary hepatocytes.     

外源硒对镉胁迫下黑麦草生长和生理的影响

为明确外源硒对重金属镉胁迫下黑麦草（Lolium multiflorum）的影响,本研究以一年生黑麦草品种"杰威"（Lolium multiflorum ‘Jiewei’）为试验材料,分析了施加0.05、0.1和1.0 mg·L^-1硒对镉胁迫（60 mg·L^-1）下黑麦草株高、根长、电导率（Electrolyte leakage,EL）、丙二醛（Malondialdehyde,MDA）含量、抗氧化酶活性及其基因表达的影响。结果表明：60 mg·L^-1镉胁迫下黑麦草株高和根长显著（P<0.05）降低,EL和MDA含量显著升高,同时超氧化物歧化酶（Superoxide dismutase,SOD）活性显著升高而过氧化物酶（Peroxidase,POD）活性显著降低,谷胱甘肽还原酶（Glutathione reductase,GR）、谷胱甘肽过氧化物酶（Glutathione peroxidase,GPX）、锰超氧化物歧化酶（Manganese superoxide dismutase,MnSOD）基因表达量显著下调,而过氧化物体抗坏血酸过氧化物酶（Peroxisomal ascorbate peroxidase,pAPX）和过氧化氢酶（Catalase,CAT）基因表达量则显著上升;施加外源硒后,镉胁迫得到缓解,在硒浓度为0.1 mg·L^-1时,株高和根长缓解效果最明显,EL和MDA降低,SOD活性下降以及POD活性上升,抗氧化酶基因表达量也恢复至对照水平,表明施加适宜浓度外源硒可调节抗氧化能力,从而维持细胞膜稳定性和有效缓解镉胁迫对黑麦草的毒害现象,其中硒浓度为0.1 mg·L^-1时效果最好。     

Effect of vitamin A and/or E on plasma enzymatic antioxidant systems and total antioxidant capacity of broiler chickens challenged with carbon tetrachloride

This study was conducted to investigate the effect of vitamin A and E supplementation on the antioxidant defences of broiler chickens against carbon tetrachloride (CCl(4))-induced oxidative stress at 4 weeks of age. Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities as well as total antioxidant (TAO) level were analysed before and after CCl(4) challenge. Day-old Lohman broiler chickens (n = 144) were randomly assigned to six factorially arranged dietary treatments consisting of vitamin A [1.35 (control) or +20 mg/kg] and vitamin E [20 (control), +40 or +60 mg/kg]. The background of vitamins A and E in the basal diet was 4500 IU (1.35 mg) and 30 IU (20 mg) respectively. At 4 weeks of age, eight chickens from each treatment were bled before interperitoneal injection with 1 ml of CCl(4) (mixed with olive oil in a ratio of 1:1) and bled again 24 h post-injection. Vitamin E supplementation decreased (p < 0.05) the activity of both SOD and GPX and showed a tendency (p = 0.07) for TAO reduction. CCl(4) attenuated SOD and GPX activities as well as TAO level. The decrease was profound (p < 0.05) in chickens fed the basal diet as well as those fed basal diet supplemented with 20 mg vitamin A. TAO levels behaved similarly when chickens were challenged with CCl(4). After CCl(4) injection, SOD activities of all experimental groups were equivalent. The presence of vitamin A decreased (p < 0.05) plasma GPX activity in chickens fed the basal diet supplemented with 40 mg/kg of vitamin E. Results of this experiment suggested that vitamin E supplementation elevated antioxidant enzyme activities while vitamin A supplementation attenuated this effect. Vitamin E supplementation improved the total reducing power by maintaining comparable levels of TAO upon CCl(4) challenge. Further experiments need to be carried out to investigate the role of vitamin A in oxidative stress and to evaluate the lipid peroxidation products.     

黄芪多糖和低浓度嗜水气单胞菌对中华绒螯蟹免疫相关因子基因表达的影响

采用逆转录-聚合酶链反应(RT-PCR)方法研究黄芪多糖和低浓度嗜水气单胞菌对中华绒螯蟹免疫相关因子基因表达的影响。结果表明:注射低浓度嗜水气单胞菌后,河蟹血细胞抗脂多糖因子(ALF)、热休克蛋白(HSP)、抗菌肽(Crus1)、脂肪酸结合蛋白酶(EsFABP3)、JAK通路细胞受体因子(EsDOME)、JAK通路信号转导与转录激活因子(EsSTAT)、河蟹肝胰腺HSP、EsFABP3和EsSTAT及河蟹鳃组织HSP的基因表达水平显著上调(P<0.05),黄芪多糖能够显著提高河蟹血细胞中ALF、酚氧化酶(PO)、HSP、Crus1、Toll样受体基因(TLR)、EsFABP3、EsJAK、EsDOME、EsSTAT、河蟹肝胰腺中ALF、PO、谷胱甘肽过氧化物酶(GPX)、HSP、TLR、EsFABP3、EsJAK、EsSTAT及河蟹鳃组织中ALF、GPX、HSP、Crus1、masquerade样蛋白(MasL)、TLR、EsJAK、EsDOME和EsSTAT的基因表达水平(P<0.05)。除了HSP和MasL外,注射黄芪多糖的河蟹免疫相关因子的基因表达水平普遍高于嗜水气单胞菌组河蟹。     

The levels of selenium and glutathione peroxidase activity in blood of sheep, cows and pigs.

The levels of selenium (Se) and glutathione peroxidase (GPX) in the blood of sheep, cows and pigs under farm conditions were examined. Sheep appear to form two distinct groups, namely high Se and GPX and low Se and GPX. The high group gave ranges of 133-249 ng/ml and 77-179 iu/g Hb for blood Se and GPX respectively, while the low group showed levels of 21-67 ng/ml and 2-20 iu/g Hb. Overall sheep blood showed a high correlation between Se and GPX (r = 0-92, P less than 0-001). Cow bloods formed one group, all having low Se and GPX levels except for a single outlier. Omitting this animal, the overall ranges were 9-72 ng/ml and 6-36 iu/g Hb for Se and GPX respectively. Blood Se and GPX activity were significantly correlated (r = 0-59, P less than 0-001). Pigs formed a single group also, with the difference that while their blood Se was high, the corresponding blood GPX activities were relatively low. Overall ranges were 93-193 ng/ml and 17-69 iu/g Hb for Se and GPX respectively. Correlation between blood Se level and GPX activity in this species was not significant (r = 0-27, P more than 0-1).     

Oxidative stress drives divergent evolution of the glutathione peroxidase (GPX) gene family in mammals

The molecular basis for adaptations to extreme environments can now be understood by interrogating the ever-increasing number of sequenced genomes. Mammals such as cetaceans, bats, and highland species can protect themselves from oxidative stress, a disruption in the balance of reactive oxygen species, which results in oxidative injury and cell damage. Here, we consider the evolution of the glutathione peroxidase (GPX) family of antioxidant enzymes by interrogating publicly available genome data from 70 mammalian species from all major clades. We identified 8 GPX subclasses ubiquitous to all mammalian groups. Mammalian GPX gene families resolved into the GPX4/7/8 and GPX1/2/3/5/6 groups and are characterized by several instances of gene duplication and loss, indicating a dynamic process of gene birth and death in mammals. Seven of the eight GPX subfamilies (all but GPX7) were under positive selection, with the residues under selection located at or close to active sites or at the dimer interface. We also reveal evidence of a correlation between ecological niches (e.g. high oxidative stress) and the divergent selection and gene copy number of GPX subclasses. Notably, a convergent expansion of GPX1 was observed in several independent lineages of mammals under oxidative stress and may be important for avoiding oxidative damage. Collectively, this study suggests that the GPX gene family has shaped the adaption of mammals to stressful environments.