肾脏铜稳态平衡调控机制研究进展

哺乳动物的生命活动过程中,几乎离不开铜和铁的氧化还原特性反应,并且铜常以Cu~(2+)的形式与其相关蛋白结合的形式作为一些功能性蛋白的辅基,同时铜结合蛋白的结构在其发挥生物学功能过程中具有重要作用。肾脏是铜排泄的器官,肾脏细胞内铜的吸收、储存、释放、转运对肾脏铜稳态很重要,肾脏铜稳态的失衡使肾脏线粒体结构发生断裂,肾脏细胞脱落导致肾脏排泄发生障碍。随着对肾脏铜转运因子与靶细胞器研究的逐步深入,论文综述了胞内的线粒体、高尔基体、胞浆分别与铜伴侣蛋白、铜转运蛋白的结合途径,阐明了铜转运蛋白(CTR1)的表达机制,铜离子转出蛋白(ATP7A)、铜离子转出蛋白(ATP7B)在高铜情况下的再分配机制,为揭示肾脏铜稳态机制奠定理论基础。     

高尿酸通过转运蛋白引起大鼠小肠黏膜上皮细胞炎性反应

研究转运蛋白(TSPO)在高尿酸引起肠上皮细胞炎症反应中的作用及机制。高尿酸体外处理肠黏膜上皮细胞,通过荧光定量PCR方法检测转运蛋白,ATP结合膜转运蛋白(ABCG2)、闭锁蛋白Occludin、上皮紧密连接相关蛋白-1(ZO-1)和IL-1β等基因表达水平,采用Western blot检测NF-κB信号通路中p65和IL-1β表达水平,采用试剂盒检测线粒体活性氧的变化,从分子水平及氧化应激反应的角度研究尿酸诱导的肠上皮细胞的炎症反应机制。最后研究高尿酸血症小鼠和正常小鼠的肠渗透性变化。结果表明,与对照组相比,高尿酸处理的肠上皮细胞TSPO、ABCG2、IL-1β等基因的mRNA的表达量显著上升(P<0.05),紧密连接相关蛋白ZO-1、Occludin表达量降低(P<0.05),p65蛋白和IL-1β的蛋白表达升高(P<0.05),活性氧(ROS)产生增高(P<0.05);高尿酸小鼠的肠渗透性明显高于正常小鼠(P<0.05)。说明高尿酸可以通过转运蛋白引起肠上皮细胞炎症反应,增加肠渗透性。     

不同铜源对鸡嗉囊ATP7A和ATP7B基因和蛋白表达的影响

为研究不同水平的硫酸铜、微米氧化铜和纳米氧化铜对鸡嗉囊铜转运蛋白基因ATP7A和ATP7B mRNA相对表达量及蛋白表达量的影响,将105只1日龄的SPF蛋鸡随机分为7组,每组15只。第1组为对照组,饲喂低铜半纯化日粮;第2组~第4组,在基础日粮中分别以硫酸铜、微米氧化铜和纳米氧化铜的形式添加铜8 mg/kg;第5组~第7组,在基础日粮中分别以硫酸铜、微米氧化铜和纳米氧化铜的形式添加铜160 mg/kg,试验期30 d。结果表明,日粮中添加硫酸铜和纳米氧化铜显著提高雏鸡嗉囊铜含量(P0.05),微米氧化铜没有明显变化。与硫酸铜和微米氧化铜相比,纳米氧化铜显著提高雏鸡嗉囊ATP7A和ATP7B mRNA和蛋白表达量(P0.05)。由此可见,嗉囊ATP7A和ATP7B对纳米氧化铜的转运发挥重要作用。     

小肽转运载体的生物学特征、影响因素及对肠道稳态的调控

小肽转运载体介导的小肽的吸收在促进动物的生长发育和提高动物生产性能中发挥着重要作用。肠道作为动物营养物质消化吸收的主要部位，肠道内环境的稳态对动物机体的健康和生长发育至关重要。由于小肽转运载体参与营养物质转运及调控肠道稳态与肠道炎症，所以肽转运蛋白成为了营养学、生理学、药理学上的研究焦点。本文就小肽转运载体的结构、转运机制、功能、表达及活性调控进行了综述，特别总结了小肽转运载体1在肠道炎症与调控肠道稳态中的作用。     

华蟾毒精对B16及RMA-S细胞主要组织相容性复合体Ⅰ类分子及其相关基因表达的影响

试验旨在考察华蟾毒精（CBG）单体对B16和RMA-S细胞MHC-Ⅰ类分子及其相关基因表达的影响。通过MTT法检测CBG对B16和RMA-S细胞生长的影响;流式细胞术检测CBG对B16和RMA-S细胞表面MHC-Ⅰ分子表达的影响;荧光定量PCR检测CBG对B16和RMA-S细胞蛋白酶体相关基因（LMP2、LMP7）和ATP结合盒（ATP-binding cassette,ABC）转运蛋白（TAP1、TAP2）基因mRNA表达的影响。结果显示,CBG对B16和RMA-S细胞MHC-Ⅰ表达的影响均不显著(P＞0.05),但高浓度CBG可不同程度提高LMP2、LMP7、TAP1、TAP2基因mRNA表达水平。结果表明,CBG可能具有通过上调LMP2、LMP7、TAP1、TAP2基因表达水平来增强肿瘤自身免疫原性的潜能。     

介导锌和铜吸收、利用的载体

载体被发现前,人们认为哺乳动物体内的锌和铜是以阴离子复合物的形式协同运输,比如金属离子与一个氨基酸结合形成螯合物或与转铁蛋白等受体结合。1995年,第一个锌离子载体(ZnT)基因ZnT1在哺乳动物中被发现。现在人们认为有两个蛋白家族参与锌的转运。ZnT蛋白家族促进胞内锌外流或内流入胞内小囊泡,降低胞内锌浓度。ZnT蛋白逆浓度梯度转运锌的机制尚不明确,然而,只有ZnT1是位于质膜上,它在动物组织中以多种方式参与锌的转运。我们实验室发现,在药理锌浓度下,ZnT1与金属硫蛋白的锌转运机制相同。第二个是Zip蛋白家族,介导细胞外液或细胞内小囊泡中的锌转运进入细胞质中,但这种蛋白却没有在家畜中发现。目前,尚未找到合适的指标来评价体内铜的状态。然而,随着近年来铜载体和伴侣蛋白的发现,研究人员认为,铜运输的调节依赖于后翻译机制介导的调节位点蛋白结构的改变。铜载体Ctr1和Ctr3,调控铜吸收的高亲和力位点。在人类的肝脏中发现一种小分子胞质蛋白MURR1,但其在铜代谢中的作用机理尚不清楚。铜伴侣蛋白可促进蛋白对铜的吸收,这可能成为衡量铜状态的一个合适的指标。研究证实,适量的高锌日粮诱导缺铜大鼠,这些大鼠组织内的Cu/Zn超氧化物歧化酶铜伴侣蛋白(CCS)含量增加,我们最近在仔猪体内也发现CCS。其他已证明的铜伴侣蛋白包括COX17和Atox1,与CCS相同,他们与细胞内的apo-酶对铜的利用有关。这些分子领域的新发现,对于评估家畜铜、锌的生物利用率和营养需要非常必要。     

胆汁酸肠肝循环转运蛋白及FXR对其的调控机制

胆汁酸作为胆汁的重要成分，由肝以胆固醇为原料进行合成，能在外源食物及相关激素的刺激下与胆汁一同被排入消化道内，具有脂肪乳化、促进肠道吸收脂质、调节肝肠功能、增加能量消耗、改善胰岛素敏感性等作用，一般可通过经典途径和替代途径两种方式进行合成。肠肝循环能将从头合成的胆汁酸重新回收约95%，仅剩余5%会流失，经替代途径进行再补充，从而保障了胆汁酸池的动态平衡，因此，肠肝循环在调节胆汁酸稳态等方面具有重要作用。近年来，随着研究的深入，胆汁酸的代谢与运输机制逐渐明确，参与肠肝循环的转运蛋白功能也更加清晰，其中，法尼酯X受体（FXR）作为重要的核因子能通过与小异二聚体受体（SHP）、视黄酸受体α（RARα）等，联合成纤维细胞生长因子15/19（FGF15/19）对胆汁酸转运蛋白的表达量进行调控，进而影响胆汁酸稳态。本文将对胆汁酸肠肝循环过程中涉及到的重要转运蛋白及FXR对其的调节机制进行阐述，为今后进一步探究胆汁酸功能提供一定的理论基础。     

肠道葡萄糖转运载体研究进展

D-葡萄糖是机体的主要能源物质,对机体代谢与内环境稳态有非常重要的作用。葡萄糖的吸收主要通过位于肠黏膜上皮细胞的两类葡萄糖转运载体家族来完成。Na+与SGLTs的结合促使载体与葡萄糖的结合,葡萄糖顺着Na+的浓度梯度进入细胞;当细胞内葡萄糖浓度升高后,葡萄糖顺着浓度差通过肠黏膜上皮细胞基底膜GLUT2经易化扩散转运进入血液。本文综述了肠道不同葡萄糖转运载体家族的成员和分类,介绍了其结构特征、功能特性及其组织分布;并详细阐述了肠道葡萄糖转运载体基因表达的影响因素。     

铜促生长作用机制

铜是畜禽必需的微量元素之一，在机体造血、新陈代谢、生长繁殖、维持生产性能。增强机体抵抗力等方面有着不可替代的作用。1945年Braude首次发现口粮中添加高铜（10倍于推荐量），可明显提高猪的生产性能，之后大量实验表明高钢可提高养猪效益（7％－32％）而得到推广。关于高铜的促生长作用机制众说纷坛，至今没有定论，似乎每种说法都有些道理。本文将铜的促生长机制归纳为两类：铜在肠道内的作用和吸收后铜在体液中的作用。1铜在肠道内的作用①铜为重金属，对蛋白质有较强的凝固作用。高铜有抑菌、抗微生物（Fuller等，1960）作用，减…     

动物肠道铁吸收、转运及其调节的分子机制

铁是动物必需的微量矿物元素,在机体内发挥氧气运输、电子传递、细胞增殖等重要的生物学功能。缺铁或铁过量均会对机体产生危害。肠道铁吸收是调节机体铁稳态的重要环节,目前已经清楚了解的铁吸收和转运机制包括肠黏膜细胞对铁的摄取、铁在细胞内的转移、铁经基底膜转运到血液并经血液循环供机体组织细胞利用等几个环节。本文拟对铁在动物体内的吸收、转运及相关调节分子机制进行综述。     

Research Progress on Regulation Mechanisms of Intestinal Copper Homeostasis

Copper is one of the necessary trace elements in animals. The absorption, storage, release and transportation of cellular copper in intestinal play an critical role in maintaining intracellular copper homeostasis, the imbalance of which can lead to intestinal dysfunction resulting from intestinal cell shedding.As for the gradually further and perfect studying on intestinal copper transporter and target organelles, people have achieved new discoveries and understanding. This article reviewed the binding pathway between intracellular mitochondria, golgi apparatus, cytoplasm and copper chaperone, as well as copper transporters. The expression mechanism of CTR1 transporters, redistribution mechanism of ATP7A and ATP7B in the case of high copper and homeostatic mechanism of other epithelial tissue copper were elucidated.Based on the above mechanism, the change of homeostasis in the intestine is a dynamic equilibrium which is related to the intestinal copper level and the expression of copper transporter in intestinal subcellular organelle,such as expression location and expression level. The conclusion will provide a theoretical foundation for revealing the mechanism of intestinal copper homeostasis.     

Age-related intestinal monosaccharides transporters expression and villus surface area increase in broiler and layer chickens

In the chicken small intestine, glucose is mainly transported by the apically located sodium/glucose cotransporter 1 (SGLT1) and the basolaterally located glucose transporter 2 (GLUT2). Fructose is transported by the apically located glucose transporter 5 (GLUT5) and similarly by GLUT2. During the early post-hatching period, the intestinal villus surface area (VSA) should be considered as an important factor related to the monosaccharide absorption capacity. Our objective here was to study intestinal monosaccharide absorption by analyzing the effects of age, diet, and breed on monosaccharide transporters and the VSA. The mRNA expression patterns of SGLT1, GLUT2 and GLUT5 genes in broiler and layer chickens were measured from the day of hatching to day 28 using the absolute quantitative real-time PCR. Both the intestinal mRNA expression levels of these genes and the VSA were affected by age. The mRNA expression levels of SGLT1 and GLUT2 were significantly increased from day 1 to day 3 and then decreased from day 3 to day 28. The expression levels of GLUT5 decreased from day 1 to day 7. The broiler chickens VSAs were significantly larger than those of the layer chickens from days 7 to 28. The effect of diet on the gene expression patterns of these monosaccharide transporters and the VSA were not significant. Our results suggest that the expression levels of these monosaccharide transporters are increased rapidly at the beginning of intestinal growth to meet the demands for monosaccharides to support the fast growth of the chick before day 7. As intestinal maturation and VSA increased, the expression levels of these monosaccharide genes decreased to a certain expression level to maintain the intestinal transport capacity and the absorption balance of all other nutrients.     

槲皮素对猪肠上皮细胞利用蛋白质的作用机制

本试验旨在研究槲皮素促进猪肠上皮细胞利用蛋白质的作用及机制.猪肠上皮细胞孵育48 h后试验组分别用含0.1、0.2、0.4、0.8和1.6 mg/L槲皮素的二甲基亚砜(DMSO)溶液处理72 h,对照组采用0.2％DMSO处理.采用二喹啉甲酸(BCA)测定受试细胞中蛋白质的含量;采用实时荧光定量PCR(RT-qPCR)...     

The Effect of Dietary Sulphate Copper Supplementation on Growth Performance, Intestinal Digestive Enzymes and Absorptive Transporters in Weanling Pigs

The experiment was conducted to assess the effects of dietary supplementation of Cu on the growth performance,digestive enzymes,tissue minerals and absorptive transporters in small intestinal mucosa of weanling pigs.One hundred crossbred pigs weaned at 28±2 d of age were assigned randomly to one of the following diets with 5 replicates:corn-soybean basal diet with 10,100,175,250 mg/kg of Cu as CuSO4·5H2O.The results showed that 250 mg/kg Cu had a positive effect (P0.05) on average daily gain,daily feed intake and ratio of gain/feed.Compared to 10 mg/kg Cu,higher Cu had significant effect on the apparent digestibility of protein and fat (P0.05).The supplementing of Cu improved amylase and lipase activity in jejunum content and lipase in pancreas (P0.05) and had no effect on intestinal morphology.The liver Cu elevated approximately 4-fold in pigs fed diet with 250 mg/kg Cu compared with pigs fed diet with 10 mg/kg Cu,no increases were observed in pigs receiving the lower level of Cu (100 and 175 mg/kg).Both Fe and Zn contents in kidney and liver were not affected by Cu supplementation.There was no positive effect (P0.05) of Cu supplementation on PepT1 (peptide transporter 1) and SGLT1 (sodium/glucose cotransporter) mRNA abundance in intestinal mucosa.However,higher supplementing level (250 mg/kg) significantly elevated the DMT1 (divalent metal transporter) mRNA abundance in duodenum mucosa.These results suggested that dietary supplementation with 250 mg/kg Cu could improve growth performance,nutrient digestibility and intestinal enzyme activities of weanling pigs.     

The Effect of Dietary Sulphate Copper Supplementation on Growth Performance, Intestinal Digestive Enzymes and Absorptive Transporters in Weanling Pigs

The experiment was conducted to assess the effects of dietary supplementation of Cu on the growth performance, digestive enzymes, tissue minerals and absorptive transporters in small intestinal mucosa of weanling pigs. One hundred crossbred pigs weaned at 28 ± 2 d of age were assigned randomly to one of the following diets with 5 replicates: corn-soybean basal diet with 10, 100, 175, 250 mg/kg of Cu as CuSO₄•5H₂O. The results showed that 250 mg/kg Cu had a positive effect (P < 0.05) on average daily gain, daily feed intake and ratio of gain/feed. Compared to 10 mg/kg Cu, higher Cu had significant effect on the apparent digestibility of protein and fat (P < 0.05). The supplementing of Cu improved amylase and lipase activity in jejunum content and lipase in pancreas (P < 0.05) and had no effect on intestinal morphology. The liver Cu elevated approximately 4-fold in pigs fed diet with 250 mg/kg Cu compared with pigs fed diet with 10 mg/kg Cu, no increases were observed in pigs receiving the lower level of Cu (100 and 175 mg/kg). Both Fe and Zn contents in kidney and liver were not affected by Cu supplementation. There was no positive effect (P > 0.05) of Cu supplementation on PepT1 (peptide transporter 1) and SGLT1 (sodium/glucose cotransporter) mRNA abundance in intestinal mucosa. However, higher supplementing level (250 mg/kg) significantly elevated the DMT1 (divalent metal transporter) mRNA abundance in duodenum mucosa. These results suggested that dietary supplementation with 250 mg/kg Cu could improve growth performance, nutrient digestibility and intestinal enzyme activities of weanling pigs.     

Effect of Copper Deficient Diet on Gut Microbiota in Mice

Copper is an essencial metal for animals to maintain fat metabolism and other important reactions.In order to study the effect of copper deficient diets on composition of intestinal microbiota and their metabolic pathways, 20 health C57BL/6 adult mice with similar body weight were chosen and fed chow diet for 7 d,and then divided into 2 groups.The mice in control group fed with copper deficient diet and 25 mg/L copper water while the experimental group mice were fed copper deficient diet and copper free water for one month.At the end of the test,the blood samples were collected to determine the ceruloplasmin content,and the caecum content were used to analyze the 16S rRNA genome by high throughput sequencing. The results showed that compared with control group,the ceruloplasmin content in experimental group was extremely significantly decreased (P < 0.001).And there were 5 siginificant differences microorganism in phylum level and 43 in genus level (P<0.05). There were 19 significant different pathways in the second grade of KEGG (P<0.05),in which metabolic-related functions were lipid metabolism, glycine biosynthesis and metabolism,metabolism of terpenoids and polyketides,biosynthesis of other secondary metabolisms and amino acid metabolism,indicating that the absence of copper in diets could alter the structure of some microbial flora and the changes of the relevant functions. It provided an important idea for studying the function of copper in the structure and metabolism of intestinal colonies in mice.     

无铜饲料对小鼠肠道菌群的影响

铜是动物机体必需的金属元素,参与重要的代谢反应。本试验旨在研究无铜饲料对小鼠肠道微生物菌落组成和相关代谢通路的影响,选择健康、体重相近、遗传背景为野生型C57BL/6J的成年公鼠20只,随机分为2组,试验组和对照组各10只。待小鼠适应性饲养1周后用无铜饲料饲喂,对照组饲喂25 mg/L含铜水,试验组饲喂无铜水,试验期30 d。试验期结束时,尾静脉采血进行铜蓝蛋白含量的测定,并采集小鼠新鲜盲肠内容物利用高通量测序技术对小鼠的盲肠内容物16S rRNA基因组进行分析。结果显示,与对照组相比,无铜组小鼠血清铜蓝蛋白含量极显著降低（P < 0.001）。无铜组小鼠盲肠微生物菌群在门、属水平分别与对照组有5和43个差异显著菌群（P < 0.05）;通过差异菌群功能预测分析发现,两组共有19种KEGG二级代谢通路差异显著（P<0.05）,其中与代谢相关的功能是脂代谢、甘氨酸生物合成与代谢、多酮类化合物和萜类化合物代谢、其他次生代谢产物的生物合成和氨基酸代谢,说明饲料中铜的缺失可以改变少数肠道菌群的组成及其功能,本试验为研究铜在小鼠肠道菌群结构及代谢中的功能提供了重要思路。