四君子汤对脾虚北京鸭胃肠组织中血管活性肠肽和生长抑素mRNA表达的影响

为从分子水平阐明脾虚与胃肠激素之间的关系以及健脾中药四君子汤防治脾虚的机制，首次用北京鸭复制脾虚证模型，以半定量的RT—PCR方法检测其腺胃、十二指肠和空肠血管活性肠肽（VIP）和生长抑素（SS）mR—NA表达的变化。结果表明，脾虚组腺胃VIPmRNA表达显著高于对照组（P〈0．05），十二指肠和空肠VIPmR—NA表达有所升高（P〉0．05）；脾虚组腺胃（P〈0．01）、十二指肠（P〈0．05）和空肠（P〈0．01）SSmRNA表达显著低于对照组。经四君子汤预防和治疗后二者恢复到接近对照组水平。提示脾虚证与VIP和SS有一定关系，四君子汤对VIP和SS水平的影响可能是该方治疗脾虚证的作用机制之一。     

慢病毒介导的生长抑素shRNA对BHK-21细胞生长的影响

采用慢病毒介导的shRNA沉默细胞自身生长抑素（SS）的表达,同时,以pcDNA3．1-SS（pSS）真核表达载体转染细胞作为阳性对照,研究SS对BHK-21细胞的抑制增殖作用,同时观察SS是否具有促进细胞凋亡的作用。MTT法绘制细胞生长曲线可知,pSS转染细胞的生长受到明显抑制,抑制效率为9．63％（P〈0．05）;而LV—sh2组细胞的生长密度是对照组细胞的117．33％（P〈0．05）,表明SS对细胞的增殖具有抑制作用。流式细胞检测细胞凋亡表明,pSS转染组和LV-shRNA感染组凋亡细胞含量分别是对照组凋亡细胞含量的1．97倍（P〈0．05）和24．30％（P〈0．05）,表明SS通过诱导细胞凋亡发挥抑制细胞增殖的作用。本研究为SS及其类似物作为治疗药物的进一步开发应用提供了理论依据。     

粘膜免疫对鸡十二指肠SS mRNA表达的影响

应用鸡新城疫弱毒疫苗进行消化道粘膜免疫，或在肌肉注射生长抑素(SS)亚单位疫苗的基础上应用鸡新城疫弱毒疫苗进行消化道粘膜免疫，通过RT—PCR方法检测免疫鸡十二指肠中SS基因的表达。结果表明，首免后第3周，新城疫免疫组的SSmRNA表达高于SS亚单位苗组，但各试验组无显著差异；首免后第4周，新城疫免疫组SSmR—NA的表达显著高于SS亚单位苗组(P＜0．05)并高于对照组。提示粘膜免疫可促进小肠粘膜SSmRNA的表达，SS亚单位苗可在基因水平上降低SSmRNA的表达。     

酸和铁胁迫对紫花苜蓿根系质膜H＋-ATPase活性的影响

以紫花苜蓿品种WL-525为材料，研究了在pH4．5和pH6．0的环境下，缺Fe^2＋和富Fe^2＋胁迫对紫花苜蓿根系质膜H^＋-ATPase活性的影响。实验结果表明：紫花苜蓿根系质膜H＋-ATPase酶的活性在强酸胁迫、缺Fe^2＋和富Fe^2＋胁迫，以及酸、铁共同胁迫下均发生明显的变化，呈现出短时间胁迫，质膜H^＋-ATPase活性升高，然后随着处理时间的增加，酶活性呈现不同程度的下降趋势。研究结果说明，在强酸和Fe^2＋胁迫下，苜蓿根系质膜H＋-ATPase酶的活性会发生变化，以减轻苜蓿根系受到的伤害。     

鸡肠道SGLT1和GLUT2 mRNA表达的组织特异性研究

运用相对定量RT-PCR方法，研究不同肠段Arbor Acre（AA）肉鸡肠道葡萄糖吸收转运主要载体SGLT1和GLUT2mRNA表达的组织特异性。结果发现。随着肠道空间位置的后移，SGLT1 mRNA的表达量逐步降低。十二指肠SGLT1 mRNA的丰度比结直肠高76．19Vo，差异极显著（P〈0．01）；而空肠和回肠SGLT1 mRNA的表达量分别比结直肠高42．86％和38．10％，差异不显著（P〉0．05），但有提高的趋势（P值分别为0．06和0．07）。十二指肠与空肠和回肠相比，SGLTlmRNA的表达量虽然分别高23．33％和27．59％，但差异不显著（P值分别为0．18和0．10）。相对定量分析表明，十二指肠和空肠GLUT2 mRNA丰度非常接近，差异不显著（P〉0．05）。定性研究显示，十二指肠与空肠GLUT2 mRNA丰度高于回肠和结直肠。鸡肠道SGLT1和GLUT2 mRNA表达的组织特异性之生理功能，有待于进一步研究。     

半胱胺在育肥猪饲养中的应用效果

在育肥猪饲料中添加不同剂量的半胱胺．探讨半胱胺对育肥猪生产性能的影响。试验选择健康的（杜X长X大）三元杂交育肥猪30头，体重58kg左右，随机分为3组。对照组饲喂基础日粮．试验1组为基础日粮＋130mg／kg半胱胺．试验2组为基础日粮＋200mg／kg半胱胺。结果表明。试验1组和2组与对照组相比。平均日增重分别提高7．13％和7．74％（P〈O．05）差异显著：料重比分别下降2．11％和2．41％（P〈O．05）差异显著；毛利收入增加6．66％和5．4％。试验1组和试验2组相比。试验2组日增重提高O．57％、料重比下降O．31％，但差异不显著（P〉0．05）；而经济收入减少1．16％。表明在育肥猪饲粮中添加130mg／kg和200mg／kg的半胱胺，均可显著提高育肥猪的日增重和饲料转化率，增加经济收入。而以添加130mg／kg半胱胺的综合效果较好。     

小鼠胚胎干细胞培养、克隆、分离、传代研究

对影响小鼠胚胎干细胞（Embryonic stem cells,ES细胞）培养、克隆、分离、传代效果的因素进行了探索研究。应用223枚昆明白小鼠胚胎和20枚129品系小鼠胚胎的研究结果表明，129品系小鼠胚胎比昆明白小鼠胚胎更适合作为ES细胞建系的材料，两者FS出现率差异显著（P＜0．05）；以DMEM＋10％NBS＋10％FCS为基础培养液，分别加入LIF、胰岛素、LIF＋SCF，极显著提高昆明白小鼠胚胎贴壁率，ICM生长率及F1、F2出现率（P＜0．01），而在DMEM＋10％NBS＋10％FCS＋LIF＋SCF为培养液，得到昆明白小鼠胚胎最高贴壁率、ICM生长率及传代率；4dpc胚胎传代情况显著好于3．5dpc胚胎（P＜0．05）。     

BOEC＋CCs、TAURIN对水牛卵母细胞体外受精及早期胚胎发育的影响

利用屠宰场废弃的水牛卵巢、观察在受精液和早期胚胎培养液中添加BOEC＋CCs(输卵管上皮细胞＋颗粒细胞）、Taurin(牛磺酸）对体外受精率及早期胚胎发育的影响。分别在受精液和早期胚胎培养液中添加BOEC＋CCs或70μM Taurin，获得的受精率分别为39．5％、40．9％，囊胚率分别为28．1％、20％，均极显著地高于对照组（P＜0．01）；同时比较发现，两实验组的受精率无明显差异，但囊胚率差异显著（P＜0．05）。     

鸡胚原始生殖细胞(PGCs)玻璃化冷冻的研究

对发育至第19期和第28期鸡胚性腺原始生殖细胞（Primordial germ cells，PGCs），用6种玻璃化冷冻液1：10％DMS0＋10％EG＋10％PVP，Ⅱ：20％EG＋10％PVP，Ⅲ：20％DMSO＋10％PVP，Ⅳ：10％DMSO＋10％EG＋0．5mol／L Surcose，Ⅴ：20％EG＋0．5mol／LSurcose，Ⅵ：20％DMSO＋0．5mol／L Surcose进行冷冻保存。结果：第19期鸡胚PGCs玻璃化冷冻复苏后存活率，在冷冻液Ⅱ与Ⅳ之间差异不显著（P〉0．05），Ⅴ与Ⅵ之间差异显著（P〈0．05），其余各冷冻液之间差异均极显著（P〈0．01）。第28期鸡胚PGCs玻璃化冷冻复苏后存活率，在冷冻液Ⅳ与Ⅴ之间差异显著（P〈0．05），Ⅲ与Ⅳ、Ⅴ之间差异不显著（P〉0．05），Ⅱ与Ⅲ、Ⅳ之间差异不显著（P〉0．05），其余各玻璃化冷冻液之间差异均极显著（P〈0．01）。复苏后接种培养传至第2代的鸡胚PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。     

果寡糖和枯草芽孢杆菌对肉鸡肠道菌群数量、发酵粪中氨气和硫化氢散发量及营养素利用率的影响

选用360只1日龄AA肉公鸡，随机分成5个处理组，每个处理设6个重复，每个重复12只鸡。试验饲粮分别为：基础饲粮（对照组），基础饲粮＋0．3％果寡糖，基础饲粮＋0．1％枯草芽孢杆菌，基础饲粮＋0．3％果寡糖＋0．1％枯草芽孢杆菌，基础饲粮＋150mg／kg金霉素（有效成分为15％）。结果表明：果寡糖和枯草芽孢杆菌具有选择性地增加肉鸡盲肠中的乳酸杆菌等有益菌群的数量，减少大肠杆菌和沙门氏菌等有害菌的数量，二者的复合添加可以更好地调节肉鸡肠道微生态环境；与对照组相比，肉鸡饲粮中果寡糖的添加使发酵粪中NH3和H2S的散发量分别降低38．38％（P〈0．05）和24．35％（P〈0．05），果寡糖＋枯草芽孢杆菌的添加使发酵粪中NH3和H2S的散发量分别降低62．14％（P〈0．05）和28．49％（P〈0．05），枯草芽孢杆菌或金霉素的添加对发酵粪中NH3和H2S的散发量均无显著影响（P〉0．05）；果寡糖、枯草芽孢杆菌和果寡糖＋枯草芽孢杆菌的添加，使肉鸡对粗灰分的利用率分别提高了18．94％（P〈0．05）、17．36％（P〈0．05）和23．66％（P〈0．05），钙的利用率分别提高了20．78％（P〈0．05）、14．63％（P〈0．05）和21．31％（P〈0．05），磷的利用率分别提高了6．60％（P〉0．05）、12．32％（P〈0．05）和14．67%（P〈0．05），但不影响粗蛋白利用率（P〉0．05）。     

Cysteamine improves growth performance and gastric ghrelin expression in preweaning piglets

The aim of the present study was to investigate the effect of cysteamine on growth performance of preweaning piglets and gastric expression of ghrelin mRNA in vivo and in vitro. Twelve litters of newborn piglets were allocated randomly to control and treatment groups. From 15 d of age, piglets in the control group were fed basal creep diet, whereas the treatment group received basal diet supplemented with 120 mg cysteamine per kg of diet until weaning on 35 d of age. Body weight gain, creep feed consumption, and diarrhea rates were recorded, and gastric mucosal tissues were collected for quantifying mRNA expression. To evaluate the direct effect of cysteamine on gastric ghrelin expression, primary cultures of gastric mucosal cells isolated from 35-d-old piglets were exposed to cysteamine for 20 h at 0, 1, 10, and 100 μg/mL, respectively. Dietary cysteamine increased (P < 0.05) average daily creep feed consumption and BW gain in preweaning pigs, which was accompanied by reduction in diarrhea rates. At 35 d of age, piglets treated with cysteamine showed increased (P < 0.05) ghrelin and gastrin and decreased (P < 0.05) somatostatin mRNA expression in gastric mucosa. Moreover, dietary cysteamine treatment increased serum concentration of gastrin (P < 0.05). In vitro, cysteamine significantly increased ghrelin mRNA expression in gastric mucosal cells at the concentration of 10 μg/mL. In conclusion, dietary cysteamine is effective in improving the growth performance and health condition of preweaning piglets, which is associated with its stimulatory effects on gastric ghrelin mRNA expression both in vivo and in vitro.     

Expression of gastric ghrelin and H(+)-K(+)-ATPase mRNA in weanling piglets and effect of ghrelin on H(+)-K(+)-ATPase expression and activity in gastric mucosal cells in vitro

This study was designed to investigate the effect of ghrelin on gastric acid secretion in weaning piglets both in vivo and in vitro. Thirty newborn piglets were selected from six litters and on 28, 35 (weaning), 38, 42 and 45d of age, respectively, one piglet from each litter was killed and the mucosal tissue from gastric fundus was collected for detecting ghrelin mRNA as well as H(+)-K(+)-ATPase mRNA expression and activity. Primary cultures of gastric mucosal cells from 5-week-old weaning piglets were challenged with 3x10(-5), 3x10(-4), 3x10(-3), 3x10(-2) and 3x10(-1)nmol/ml h-ghrelin, respectively, for 4h in order to further clarify the effect of ghrelin on gastric H(+)-K(+)-ATPase mRNA expression and activity. Ghrelin mRNA expression in gastric fundus kept stable from 28d to 42d, followed by a sudden increase on 45d, exhibiting a peak that was significantly higher than any other age groups investigated. H(+)-K(+)-ATPase activity and mRNA expression showed similar trends of increase with slightly different timing. H(+)-K(+)-ATPase mRNA expression tended to increase on 42d, while H(+)-K(+)-ATPase activity started to rise from 35d, but neither of them reached significantly higher levels until 45d. In vitro, ghrelin significantly increased H(+)-K(+)-ATPase activity of gastric mucosal cells at 3x10(-4), 3x10(-3), and 3x10(-2)nmol/ml, but augmented H(+)-K(+)-ATPase mRNA expression only at 3x10(-4)nmol/ml. The results indicate that ghrelin mRNA expression is up-regulated 10 days after weaning in the gastric fundus of piglets, coinciding with the increase of H(+)-K(+)-ATPase mRNA expression and activity. Ghrelin acts on gastric mucosal cells to stimulate both mRNA expression and activity of H(+)-K(+)-ATPase in vitro.     

黏膜免疫和半胱胺对十二指肠生长抑素mRNA表达的影响

应用鸡新城疫弱毒苗单一口服和分别灌喂两种剂量半胱胺后再口服鸡新城疫弱毒苗,进行消化道黏膜免疫肉鸡,通过RT-PCR方法检测十二指肠中SS基因表达。结果表明,首免第4周后各个试验组SSmRNA的表达无显著差异。首免第5周后两种剂量半胱胺结合新城疫免疫组SSmRNA的表达比对照组显著增加(P<0.05),单一新城疫免疫组SSmRNA的表达虽然比对照组增加,但并不显著。提示黏膜免疫后淋巴细胞产生的细胞因子可刺激十二指肠SSmRNA的表达,半胱胺可增加十二指肠SSmRNA的表达。肠黏膜免疫可影响动物体内收稿日期:2002-06-17课题来源:国家自然科技基金(30070564)作者简介:杨　倩(1962-),女,南京农业大学动物医学院副教授,博士,主要从事动物免疫神经内分泌研究工作。神经内分泌系统中SSmRNA的表达。     

新型饲料添加剂——半胱胺的作用机理及其对动物生产性能的影响

内源半胱胺 (cysteamine ,CS)在动物体内主要产生于半胱氨基酸的代谢 ,含量很低。添加外源CS能迅即选择性地降低动物体内生长抑素 (somatostatin ,SS)的免疫活性与生物活性。CS主要通过这种机理来解除SS对动物生长的抑制 ,提高GH的基线水平和其它受SS抑制的有关激素的水平 ,加强消化道的蠕动和消化酶的分泌 ,从而CS处理能起到促进动物生长和改善生产性能的作用。近年来研究表明CS处理能明显提高生长期动物的生长 ,并且能提高泌乳动物的泌乳性能。本文综述了近年来CS的研究和应用方面的进展。     

粘膜免疫对鸡十二指肠SS mRNA表达的影响

应用鸡新城疫弱毒疫苗进行消化道粘膜免疫,或在肌肉注射生长抑素(SS)亚单位疫苗的基础上应用鸡新城疫弱毒疫苗进行消化道粘膜免疫,通过RT PCR方法检测免疫鸡十二指肠中SS基因的表达.结果表明,首免后第3周,新城疫免疫组的SS mRNA表达高于SS亚单位苗组,但各试验组无显著差异;首免后第4周,新城疫免疫组SS mRNA的表达显著高于SS亚单位苗组(P＜0.05)并高于对照组.提示粘膜免疫可促进小肠粘膜SS mRNA的表达,SS亚单位苗可在基因水平上降低SS mRNA的表达.     

Pharmacokinetics of amoxicillin after oral administration in recently weaned piglets with experimentally induced Escherichia coli subtype O149:F4 diarrhea

OBJECTIVE: To measure the effect of Escherichia coli subtype O149:F4-induced diarrhea on the pharmacokinetics of orally administered amoxicillin in affected piglets relative to that of uninfected piglets. ANIMALS: 22 healthy 4-week-old recently weaned Danish crossbred piglets. PROCEDURE: 12 piglets were orally inoculated through gastric intubation with 10(9) CFUs of an E. coli O149:F4 strain and responded by developing diarrhea 12 to 16 hours later. Piglets were dosed with amoxicillin trihydrate solution (20 mg/kg) by gastric intubation. A control group of 10 age-matched piglets without signs of diarrhea was dosed similarly. Blood samples were obtained before amoxicillin administration and at 0.5, 1, 1.5, 2, 3, 6, 12, and 24 hours after amoxicillin administration. The plasma concentration of amoxicillin was analyzed by high-performance liquid chromatography. RESULTS: A significant 39% decrease in the area under the plasma concentration versus time curve of amoxicillin was observed in piglets with diarrhea relative to that of control piglets. The maximum plasma concentration (Cmax) was significantly (52%) lower in piglets with diarrhea, compared with control piglets, while the elimination rate constant, time to reach Cmax, and elimination half-life were unchanged. CONCLUSIONS AND CLINICAL RELEVANCE: Escherichia coli-induced diarrhea may decrease systemic bioavailability of amoxicillin. Escherichia coli bacteria attach to the intestinal epithelial cells. Because it is assumed that the concentration of the antimicrobial at the site of infection reflects the systemic concentration, higher doses of amoxicillin in the treatment of piglets with E. coli O149:F4-induced diarrhea may be appropriate.     

荷斯坦牛血红细胞Na~+-K~+-ATP酶活力与其耐热性的相关性研究

[目的]为探讨Na+-K+-ATP酶在荷斯坦牛热应激过程中的作用.[方法]本研究比较测定了329头荷斯坦牛在非热应激期和热应激期的体温、红细胞K+和红细胞Na+-K+-ATP酶活力的变化.[结果]显示,试验牛群红细胞Na+-K+-ATP酶活力呈正态分布;不同胎次、产犊季节、泌乳期、场次牛Na+-K+-ATP酶活力差异不显著(P>0.05);不同性别牛Na+-K+-ATP酶活力差异极显著(P<0.01).在热应激期,同一泌乳期牛红细胞Na+-K+-ATP酶活力与产奶量下降率成负相关;荷斯坦牛的体温明显升高,产奶量明显下降,红细胞K+无显著变化,红细胞Na+-K+-ATP酶活力明显下降,与非热应激期相比只有红细胞K+差异不显著(P>0.05),其余都差异极显著(P<0.01);红细胞Na+-K+-ATP酶与体温成负相关,与产奶量成正相关,与红细胞K+的相关性不显著.[结论]在热应激期,荷斯坦牛红细胞Na+-K+-ATP酶活力与耐热性成正相关,红细胞Na+-K+-ATP酶活力高的荷斯坦牛,耐热性好.     

甘氨酸对脂多糖刺激仔猪肝脏能量代谢及相关基因表达的调控作用

本试验旨在研究甘氨酸(Gly)对脂多糖(LPS)刺激断奶仔猪肝脏能量代谢、能量代谢关键酶和相关调节因子mRNA表达的调控作用。选取24头杜×长×大仔猪,分成4个组,每组6个重复。4个组分别为:1)对照组(基础饲粮);2)LPS组(LPS+基础饲粮);3)1.0%Gly组(LPS+基础饲粮+1.0%Gly);4)2.0%Gly组(LPS+基础饲粮+2.0%Gly)。试验第28天,试验组仔猪腹膜注射100μg/kg BW的LPS,对照组注射等量的生理盐水。试验猪于注射LPS或生理盐水4 h后屠宰,取肝脏样品待测。结果表明:1)与LPS组相比,1.0%Gly显著提高了仔猪肝脏三磷酸腺苷(ATP)浓度和能荷(EC)水平(P0.05),显著降低了一磷酸腺苷(AMP)/ATP值(P0.05),并有降低AMP浓度的趋势(P0.10);2.0%Gly有降低AMP/ATP值的趋势(P0.10)。2)与LPS组相比,1.0%Gly显著降低了仔猪肝脏己糖激酶2(Hexok2)和柠檬酸合成酶(CS)的mRNA表达量(P0.05);2.0%Gly显著降低了肝脏Hexok2和丙酮酸激酶(PK)的mRNA表达量(P0.05),并有降低肝脏CS mRNA表达量的趋势(P0.10)。3)与LPS组相比,1.0%Gly显著提高了仔猪肝脏腺甘酸活化蛋白激酶α1(AMPKα1)的mRNA表达量(P0.05)。综上所述,饲粮中添加Gly能够改善LPS刺激引起的断奶仔猪肝脏能量代谢紊乱,调控糖酵解和三羧酸循环等代谢途径中相关酶的表达。     

Responses of plasma Epo and kidney and liver Epo mRNA to hemorrhage in perinatal pigs

Despite the fact that pig fetuses in late gestation have extensive erythropoiesis, low blood pO₂ and low hemoglobin concentrations, piglets are born without detectable concentrations of plasma erythropoietin (Epo). In the present study, we have examined the hypothesis that long-term hypoxic stimuli are less efficient than short-term stimuli in stimulating Epo production in perinatal pigs. From fetuses collected by hysterectomy 5 days before term, new-born piglets and piglets 2 and 5 weeks old, blood in amounts corresponding to 2% of body weight was withdrawn from the jugular vein. Twenty-four hours later the animals were killed and their kidney and liver Epo mRNA analysed by a competitive RT-PCR assay. Plasma Epo concentration was estimated by a solid-phase, two-site sequential chemiluminescent enzyme immunometric assay. We found that in nearly fully developed fetuses and in new-born piglets, the concentration of Epo mRNA did not increase upon bleeding. This is in contrast to earlier findings in sheep. In 2- and 5-week-old piglets, bleeding was associated with a 12–15-fold increase in kidney Epo mRNA. In the 2- and 5-week-old piglets, bleeding evoked increased translation of Epo mRNA into the protein hormone. Also in new-born piglets, increased plasma levels of Epo accompanied bleeding, whereas significant changes in gene Epo expression were not observed.     

生长抑素基因疫苗pcS/SS构建及其在HeLa细胞中的表达

将 PCR扩增得到的 pc MV- S中的乙肝表面抗原 (HBs Ag) S基因亚克隆到 p UC18中 ,构建成 p U S质粒 ,再将化学合成的生长抑素 (somatostatin,SS)基因单链复性 ,融合到 S基因编码区的第 2 2 5个氨基酸位点之后 ,构建成 p US/SS质粒 ,然后将 S/ SS融合基因亚克隆到 pc DNA3.1(- )中 ,构建成 S/ SS融合表达质粒 pc S/ SS。采用脂质体包裹法将 pc S/ SS质粒转染 He L a细胞 ,用 SDS- PAGE和 EL ISA分析表明 ,S/ SS融合基因在转染后 72 h和 G4 18选择 2周后的细胞中均获得表达 ,融合蛋白具有 SS的反应原性。