铜对肉鸡原代肝细胞活性及糖原含量的影响

为了探讨铜对肉鸡原代肝细胞活性及糖原含量的影响,试验分别用10,50,100 μmol/L Cu2+在体外孵育原代肝细胞,PAS染色后测定30分钟和1,2,4,24,48,72小时各时间点的原代肝细胞活 性及24小时时的糖原含量.结果表明:50 μmol/L和100 μmol/L Cu2+仅分别在72小时和4小时...     

维生素C对铜孵育的肉鸡原代肝细胞周期及细胞膜电位的影响

为了研究维生素C对铜孵育的肉鸡原代肝细胞周期及细胞膜电位的影响,试验将全量换液24 h后的原代肝细胞分为正常对照组(Ⅰ组)和10μmol/L Cu2+(Ⅱ组)、30μmol/L Cu2+(Ⅲ组)、50μmol/L Cu2+(Ⅳ组)、50μmol/L维生素C+50μmol/L Cu2+(Ⅴ组)刺激组,于体外刺激后48小时用流式细胞仪测定各组的细胞周期及细胞膜电位。结果表明:30μmol/L和50μmol/L Cu2+能明显引起静止期肝细胞数增多,增殖期肝细胞数减少,细胞膜电位下降(P<0.05),10μmol/L Cu2+组没有出现明显变化。说明维生素C可显著抑制50μmol/L Cu2+引起的变化,对细胞具有保护作用。     

铜对猪原代肝细胞含铜酶活性的影响

选用新生仔猪的肝进行细胞分离培养,建立肝细胞体外培养模型,施以不同质量浓度的铜进行试验,观察记录肝细胞生长情况,检测含铜酶中的铜蓝蛋白(CP)、铜锌超氧化物歧化酶(CuZn-SOD)和丙二醛(MDA)的变化.试验结果表明:在细胞培养液中添加一定质量浓度的铜,可提高CP和CuZn-SOD的活性.低质量浓度的铜对CP的影响很小;当铜质量浓度逐渐升高时,CP活性显著升高,但当铜质量浓度过高则抑制CP活性.低质量浓度铜对MDA影响不大,但随着铜质量浓度的增加MDA含量迅速升高.     

铜对猪原代肝细胞中铜锌超氧化物歧化酶和丙二醛活性的影响

肝细胞的体外培养是研究肝脏和肝细胞功能的有效途径。虽然体外培养的肝细胞不受完整机体所存在的神经体液调节作用的影响,但由于其便于施加各种试验因素,从而用于多方面的研究。试验拟在建立体外培养原代肝细胞模型的基础上,通过向体外培养的肝细胞模型中添加铜,观察肝细胞中铜锌超氧化物歧化酶(CuZn-SOD)和丙二醛(MDA)的活性变化,为探究高铜促生长机理提供试验依据。1材料与方法1.1材料1.1.1试验动物选用初生1~3日龄的“军牧1号”健康仔猪作为肝细胞供体,雌雄不限,体重0.5~1.0 kg,由吉林大学农学部大学动物繁育中心种猪场提供。1.1.2…     

铜对肉鸡原代肝细胞三磷酸腺苷水平的影响

本试验以五水硫酸铜作为试验铜源,设0μmol/L、50μmol/L、100μmol/L、150μmol/L四个浓度组。采用荧光酶标仪,分别在4 h、8 h、12 h、24 h和48 h检测肉鸡原代肝细胞内各组三磷酸腺苷(ATP)水平的变化情况。结果 48 h时50μmol/L组ATP水平最高,之后逐步降低;100μmol/L组和150μmol/L组在8 h时ATP水平升高,之后逐步降低。表明铜的浓度越高、作用时间越长,细胞ATP水平则越低。     

肉鸡原代肝细胞对铜的摄入及铜对胞内钙超载作用

为探讨肉鸡原代肝细胞对铜的摄入及铜对胞内游离钙浓度的影响,将全量换液24 h后的细胞分为空白对照组(Ⅰ组)和10μmol/L Cu~(2+)(Ⅱ组)、50μmol/L Cu~(2+)(Ⅲ组)、100μmol/L Cu~(2+)(Ⅳ组)试验组,于体外培养48 h后,采用火焰原子吸收光谱法测定每组细胞外液的Cu~(2+)含量,并观察不同剂量Cu~(2+)作用下胞内游离钙的变化。结果表明,试验组细胞Cu~(2+)的吸收量均超过85%。各试验组均能明显引起肝细胞内游离钙浓度升高,与空白对照组比较差异显著(P0.05),且胞内游离钙浓度随Cu~(2+)剂量的增大而升高。结果显示,Cu~(2+)进入了体外培养的肉鸡肝细胞,且Cu~(2+)对胞内游离钙浓度的影响有明显剂量依赖性,Cu~(2+)浓度越高,造成胞内游离钙超载。     

铜导致肉鸡肝细胞游离钙的超载

选用硫酸铜作为试验铜源,向即时分离的肉鸡肝细胞悬液中加入不同剂量的铜(铜终浓度分别为:0,5,10,20,30μmol/L),孵育不同时间(5,10,15,20,30 min)后观察肝细胞内游离钙浓度的变化.结果显示:高剂量的铜可破坏细胞内游离钙稳态.钙内流造成细胞内钙超载,30μmol/L铜刺激组的[Ca2+]i显著高于对照组(P<0.05).肝细胞在10 μmol/L Cu2+环境中孵育不同时间后显示,时间越长.细胞内钙浓度越高;此结果表明,10μmol/L铜能诱导细胞内游离钙的超载,并具有浓度依赖性和时间依赖性,暗示铜对体细胞的毒害作用可能是通过改变细胞内游离钙浓度来实现.     

镉对原代培养大鼠肝细胞的损伤作用

用活体肝脏灌注、胶原酶消化法分离大鼠肝细胞并作原代培养,以CdCl2(0～250或500umol/L）处理,测定培养上清液中LDH、ALT、ARG和细胞内GSH-Px、GSH、MDA等的含量,探讨镉对肝细胞的损伤作用和脂质过氧化状况。结果当Cd≥10和5umol/L时,细胞内LDH、ARG泄漏分别明显增加,GSH-Px活力显著受到抑制,GSH含量和MDA产生随着镉的剂量增大而增加（Cd≥50 umol/L）。认为镉对肝细胞的损伤和对细胞膜完整性的破坏,与脂质过氧化有关;镉处理后细胞中GSH增加可能与镉抑制了还原型谷胱甘肽向氧化型谷胱甘肽的转化有关。镉可直接抑制ALT本身的活性,因而认为以细胞内ALT泄漏作为镉诱导细胞损伤的评价指标时要予以注意。     

铜对大鼠肝细胞活性、自由基代谢及细胞膜电位的影响

通过建立大鼠肝细胞的铜胁迫模型,旨在讨论不同剂量的铜对大鼠肝细胞活性、自由基代谢及细胞膜电位的影响。以硫酸铜为试验铜源,6个不同铜质量浓度(以Cu2+计):0,50,100,200,400,800mg/L,分别加入培养液培养BRL-3A细胞24和48h后,测定不同Cu2+质量浓度、不同处理时间后细胞自由基产生、抗氧化酶活性及细胞膜电位变化。结果显示,处理质量浓度低于400 mg/L时,细胞增殖活性差异不显著(P0.05),处理质量浓度为400,800mg/L时,细胞活性下降(P0.05);处理质量浓度低于400 mg/L时,Cu2+质量浓度与细胞内T-SOD、GSH-Px、CP活性呈正相关,与MDA、NO含量呈负相关,质量浓度达400mg/L及以上质量浓度时,结果相反。随着Cu2+质量浓度的增加,细胞膜电位下降(P0.05)。以上结果表明,适量的铜含量对细胞内的抗氧化酶活性具有促进作用,但高铜可能促使细胞活性下降,自由基产生增多,细胞膜电位下降。     

原代牛肝细胞分离和培养方法的建立

采用胶原酶灌注消化法,对来自新生小牛血清制备厂的牛肝脏尾状突材料进行肝细胞分离,用台盼蓝拒染法测定总细胞数和肝细胞即时存活率,以每孔1×106个肝细胞的量接种于牛尾胶原包被的6孔细胞培养板,进行单层培养细胞形态学观察和培养基上清液LDH活性测定.结果显示,每头小牛肝脏尾状突分离获得的细胞产量为(3.558±0.096)×108个,肝细胞即时存活率为(84.46±3.56)%,培养24～72 h的肝细胞生长状态最好,适合用于有关肝细胞代谢、中毒、基因表达的研究.     

硼对铜中毒肉用乌骨鸡肝铜含量及肝功能的影响

选用560只1日龄肉用乌骨鸡,采用二因子多水平随机化试验设计研究日粮硼对铜中毒肉鸡肝铜及肝功能的影响。分别在玉米-豆粕型基础日粮中(Cu50.9 mg/kg,B10.1 mg/kg)添加549.1、749.1 mg/kg的铜和49.1、109.1 mg/kg的硼。结果表明,硼可在一定程度上抵御高铜对肝细胞的毒害,并减少肉鸡肝铜的蓄积量,说明硼对肉用乌骨鸡铜中毒具保护效应。     

高铜对雏鸡器官组织病理损害的研究

将420只1日龄艾维菌健雏，随机分为7组，分别饲以含铜量11mg／kg的基础日粮和含铜量分别为100、200、300、400、500和600mg／kg的6种高铜日粮6周，研究高铜对雏鸡的病理损害。结果显示，日粮含铜100和200mg／kg对雏鸡有促生长作用。日粮铜水平达300mg／kg以上时，对雏鸡器官组织造成不同程度的病理损伤，表现为肌胃角质层增厚呈黄褐色；肝呈浅黄色或深褐色，肝细胞脂肪变性；消化道上皮细胞变性坏死；免疫器官和肾、心脏、胰腺、大脑等器官的实质细胞肿胀；淋巴免疫器官体积缩小，淋巴细胞数量减少。超微结构观察发现，肝细胞胞浆和胞核内见有数量不等、大小不一的高电子密度的沉积物；凋亡的淋巴细胞染色质呈花瓣样、半月形或环形，聚集在核膜下。表明，肝、消化道和免疫器官是高铜损害的主要靶器官。     

谷胱甘肽对铜孵育的肉鸡肝细胞线粒体的保护作用

向即时分离的肉鸡肝细胞线粒体培养液内加入终浓度分别为10、30、50μmol/L的Cu2＋和200μmol/L GSH＋50μmol/L Cu2＋,孵育20min后,观察培养液中不同铜浓度对线粒体H2O2生成速率和呼吸链复合物活性的影响。结果表明,30μmol/L和50μmol/L的Cu2＋能引起线粒体H2O2生成速率和呼吸链复合物活性出现明显加快和降低（P〈0.05或P〈0.01）,10μmol/L Cu2＋组没有出现明显变化;谷胱甘肽可显著抑制50μmol/L Cu2＋引起的变化（P〈0.01）,对线粒体具有保护作用。     

高铜对肉鸡原代肝细胞周期及细胞膜电位的影响

通过流式细胞仪测定各组的细胞周期及细胞膜电位,研究高铜对肉鸡原代肝细胞凋亡的影响。将全量换液24h后的细胞分为空白对照组和低铜组（10μmol/L Cu2＋组）、高铜Ⅰ组（50μmol/L Cu2＋组）、高铜Ⅱ组（100μmol/L Cu2＋组）,培养48h后测定荧光值,结果表明,高铜组能显著引起静止期（G0/G1）肝细胞数增多、增殖期（S、G2＋M）肝细胞数减少、细胞膜电位下降（P〈0.05）;低铜组未出现明显变化;表明高浓度的铜能明显改变肝细胞的细胞周期及细胞膜电位。     

The effect of copper deficiency on the peripheral blood cells of cattle

Six female cattle were given molybdenum (30 ppm) and sulphate (225 ppm) to induce experimental secondary copper deficiency. The total and differential leukocyte numbers and lymphocyte subpopulations were counted and the neutrophil activity was assessed by means of nitroblue tetrazolium reduction and phagocytosis of sheep erythrocytes. The serum caeruloplasmin activity and concentration were also determined. Copper deficiency was confirmed from decreased serum copper levels, the animals with values less than 5.9 µmol/L being considered deficient. Total leukocyte numbers were not affected by the copper deficiency. However, differential counts showed a marked increase in monocyte subpopulations, a significant decrease in B lymphocytes and reduced neutrophil activity. The serum caeruloplasmin activity was decreased about 50%, but the total serum protein concentration was less altered. We concluded that the effect of these changes on the animals' immune competence may contribute to a greater incidence of infectious diseases in copper-deficient cattle.     

The effects of levamisole on oxidative stress induced by copper intoxication in broilers

Abstract

AIM: To determine the effects of Cu and levamisole on concentrations of Cu and Fe in plasma and liver, and the effects of levamisole on lipid peroxidation induced by Cu intoxication in broiler chickens.

METHODS: In a 2×4 factorial study, 80 one-day-old Ross PM3 broiler chicks were fed diets for 21 days containing either 8 mg/kg Cu (Low Cu) or 250 mg/kg Cu (High Cu) and were treated with 0 (L0), 4 (L4), 8 (L8) or 16 (L16) mg/kg bodyweight levamisole per day from Day 7 of the study, on three consecutive days in their drinking water. This treatment was repeated three times, at 3-day intervals. On Day 21, blood samples were collected from each bird for analysis of concentrations of Cu, Fe and malondialdehyde, and activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), superoxide dismutase, catalase and glutathione peroxidase (GSH-Px). The birds were killed and liver samples collected for analysis of Cu and Fe.

RESULTS: Mean concentrations of Cu and Fe in plasma, and Cu in liver, were increased overall in the High Cu groups compared with the Low Cu groups (p<0.001). Compared with the L0 treatment group on the High Cu diet, treatments L4, L8 and L16 decreased concentrations of Cu in plasma, and L8 and L16 increased concentration of Cu in liver (p<0.05). Mean activities of AST and ALT were increased in untreated birds (L0) fed the High compared with Low Cu diets (p<0.01). In birds receiving the High Cu diet, treatments L4 and L8 decreased activities of AST, and L4 and L16 decreased activity of ALT, compared with L0 (p<0.05). The High Cu diet induced an oxidative stress characterised by increased mean concentrations of malondialdehyde and decreased activities of superoxide dismutase, catalase and GSH-Px (p<0.001). Concentration of malondialdehyde, and activities of superoxide dismutase and catalase were not changed following levamisole treatment in birds on the High Cu diet, and activity of GSH-Px was decreased by the L4 and L8 treatments compared with the L0 group.

CONCLUSIONS AND CLINICAL RELEVANCE: The results of the study suggest that treatment with levamisole might alleviate the harmful effects of Cu on the liver, as demonstrated by decreased activities of AST and ALT induced by a diet containing 250 mg/kg Cu.     

福美双和铜对肉鸡胫骨软骨发育不良的影响

本试验旨在研究福美双和铜对肉鸡胫骨软骨发育不良、血清相关酶活性和组织铜含量的影响。100羽1日龄AA肉鸡随机分为4组,从8日龄起分别给予基础日粮(对照组),福美双日粮(基础日粮+福美双100 mg/kg),福美双低铜日粮(基础日粮+福美双100 mg/kg+CuSO4.5H2O 50 mg/kg),福美双高铜日粮(基础日粮+福美双100 mg/kg+CuSO4.5H2O 100 mg/kg),试验期至21日龄。结果表明:(1)日粮中添加福美双(100 mg/kg)能显著增加肉鸡TD的发生率;添加铜能降低福美双的这种作用,两者之间存在明显的互作关系;(2)与对照组相比,福美双组和福美双高铜日粮组肉鸡血清钙、磷水平明显升高(P<0.05);血清ALT水平在各组之间无显著差异;血清ALP、AST水平,福美双高铜日粮组与其余各组相比明显升高,差异显著(P<0.05);(3)与对照组相比,其余各组肉鸡胫骨长度均显著下降(P<0.05);(4)日粮中铜添加可提高各组织中铜的沉积;添加福美双能提高肝脏、肾脏、胰腺中铜的沉积,同时降低铜在肾脏中的沉积,而铜的添加能够降低福美双所引起的肾脏铜的增加。提示福美双可能通过损伤肝、肾功能,影响机体组织对铜的生物利用从而诱导肉鸡胫骨软骨发育不良的发生。     

日粮不同铜、维生素A水平及互作效应对肉仔鸡体内几种蛋白代谢相关指标的影响

选用1日龄艾维因肉仔鸡448只,随机分为8组,每组4个重复,每重复14只(公母各半).用4×2(Cu×VA)完全随机设计,铜的添加量为0、8、150、225 mg/kg,维生素A的添加量为1 500和5 000 IU/kg.探讨日粮不同铜、维生素A水平及互作效应对肉仔鸡体内几种蛋白代谢相关指标的影响.结果显示日粮低铜(0～8 mg/kg)组血清尿素氮(SUN)质量浓度降低,血清总蛋白(TP)质量浓度提高,血清谷草转胺酶(GOT)和谷丙转胺酶(GPT)的活性提高;高剂量维生素A(5 000 IU/kg)组SUN质量浓度降低,TP质量浓度显著增加;低铜(0 mg/kg)与高剂量维生素A(5 000 IU/kg)在降低SUN质量浓度和提高TP质量浓度方面有协同作用.结果表明低铜(0～8 mg/kg)利于肉仔鸡体内蛋白质合成.     

The effect of copper sulphate on performance and the structure of the gizzard in broilers

Addition of copper sulphate (about 200 mg per kg) to conventional diets produced a small, though variable, growth response and improved food conversion in broilers maintained under commercial conditions. In some experiments interactions were found between sex or coccidiostat and copper in the diet. Broilers fed on diets containing greater concentrations of copper (between 400 and 600 mg per kg) exhibited reduced growth rate and food intake. The gizzard linings of birds receiving diets containing 600 mg CuSO₄ per kg showed extensive damage. This damage was attributable to a massive increase in the shedding of the gizzard glandular cells into the koilin layer and to disruption and cessation of koilin production. Similar though less severe gizzard changes occurred in birds which received 400 mg per kg and there was a slight increase in cellular desquamation in some of the birds receiving 200 mg per kg. High concentrations of copper were found in the gizzard lining and caecal contents of birds receiving supplementary copper and there was a progressive increase in the concentration of copper in the litter on which such birds had been reared.