【作者】 虞泽鹏；

【导师】 乐国伟； 施用晖；

【作者基本信息】 江南大学 ， 粮食、油脂与植物蛋白工程， 2005， 博士

【摘要】 锌是动物的一种必需微量元素,对维持动物生长、发育和免疫功能起着重要的作用。氨基酸螯合锌是锌同氨基酸按一定摩尔比以共价键结合而成有机微量元素螯合物;有研究表明其能有效促进动物生长和提高机体免疫;亦有研究认为两者被动物吸收后在体内的代谢不同。本课题采用肉用仔鸡和小鼠作为试验对象,选用蛋氨酸锌（zinc methionine,Zn-Met）和硫酸锌（ZnSO₄）两种锌源研究两者对动物生长、免疫的调节效果及其分子机制。1.不同锌源对肉用仔鸡早期生长的影响及DDRT-PCR法初步筛选差异表达基因选用1日龄Avine肉用仔鸡75只,随机分为3组（对照组,ZnSO₄组和Zn-Met组）,每组5个重复。测定试鸡生长性能及部分生理生化指标,研究不同锌源对仔鸡早期生长的影响;同时采用银染差异显示技术（differential display reverse transcription PCR,DDRT-PCR）进行差异表达基因的初步筛选。结果表明,基础日粮中添加Zn-Met和ZnSO₄均能有效提高仔鸡生长和改善饲料效率;Zn-Met组效果优于ZnSO₄组。添加锌能提高胫骨、胰脏中锌含量;Zn-Met组锌增加幅度高于ZnSO₄组。血清锌含量及血清AKP活性三组间无显著差异。试验采用银染差异显示法对受不同锌源影响肝脏基因进行了初步筛选,回收差异条带26条;对其中两个差异条带进行亚克隆,成功获取一个阳性克隆。2.不同锌源对断乳小鼠早期生长的影响及其机制选取21d断奶雄性小鼠（KM种）90只,随机分为3组,每组设3个重复,采用玉米淀粉—酪蛋白半纯合日粮基础日粮（含锌11.67mg/kg）。对照组饲喂基础日粮,硫酸锌组、蛋氨酸锌组分别在基础日粮中添加ZnSO₄、Zn-Met 30mg/kg（以锌计）;测定小鼠体重、组织锌含量、血清AKP活性、血浆生长激素（GH）含量、肝脏生长激素受体（GHR）及胰岛素样生长因子I（IGF-I）mRNA表达水平。结果表明,两种形式锌均能有效提高小鼠体重（P<0.05）;蛋氨酸锌促长效果优于硫酸锌,在饲养前期尤为显著（P<0.05）。两种锌源均能提高肝脏、血清锌含量及血清AKP活性;蛋氨酸锌处理组织锌含量增加幅度略高于硫酸锌处理（P>0.05）。两种锌源对GH水平、GHR基因表达无显著影响（P>0.05）;均能显著提高IGF-I基因表达水平（P<0.05）。与ZnSO₄相比,Zn-Met更大幅度地提高IGF-I mRNA表达（P<0.05）,从而有效地促进小鼠生长（P<0.05）。3.不同锌源吸收、转运及调节机制研究选用21d断奶雄性小鼠为研究对象,半定量RT-PCR法测定小肠ZnT1（zinctransporter,ZnT）、ZnT4、DCT1（divalent cation transporter,DCT）、小肠及肝脏MT-1（metallothionein,MT）mRNA表达量。结果表明,Zn-Met、ZnSO₄处理显著降低ZnT1、ZnT4、DCT1 mRNA表达量（P<0.05）;Zn-Met组ZnT1、DCT1 mRNA表达量均显著低于ZnSO₄组（P<0.05）;两种锌处理间ZnT4 mRNA表达量无明显差异（P>0.05）。小鼠肝脏、小肠MT-1 mRNA表达量均为Control<ZnSO₄<Zn-Met（P<0.05）。上述结果表更多还原

【Abstract】 Zinc is an essential trace-element required for growth, development and immunity ofanimals. Growth retardation and immunity defect are principal signs of zinc deficiency inhuman and animals, but the underlying mechanisms remain to be elucidated. Zinc methionine（Zn-Met） has been shown to have greater bioavailability than inorganic zinc sources in somesituations. The aim of our study was to evaluate the effects and its molecular mechanism ofzinc sulfate and zinc methionine on growth, and immunity in broilers and mice.1. Effect on production performance on early stage of broilers and differential display ofhepatic mRNAs regulated by different forms of zinc in dietsThis experiment was conducted to investigate the effect of different forms of zinc onproduction performance and to study the mechanism of different forms of zinc on animals bydifferential display reverse transcription PCR （DDRT-PCR）. Seventy-five Avine broilers wereallotted at random into 3 treatments. The control was fed on corn-soybean basal diet.Treatments of ZnSO₄ and Zn-Met were supplemented with zinc sulfate and zinc-methionine at40 mg/kg zinc. The results showed that supplementing zinc in basal diet enhanced weight gainof 14, 21 day and improve feed conversion efficiency;Zn-Met showed more effectively thanZnSO₄. Zinc contents in tibia and pancreas were increased by zinc supplement;they wereincreased more by Zn-Met as compared to ZnSO₄. There were no significant difference ofserum zinc content and AKP activity among the three treatments （P>0.05）. Twenty-sixdifferent bands were obtained from DDRT-PCR. Two of the differential fragments of cDNAswere sub-cloned;one positive clone was received successfully.2. Effect of zinc sulfate and zinc methionine on growth and their mechanism in miceNinety male KM mice were randomly divided into three treatments to investigate the effectof zinc sulfate and zinc methionine on growth and their possible regulating mechanism. Thecontrol was fed on the basal diet containing zinc of 11.67mg/kg. The treatments of ZnSO₄ andZn-Met were fed on the diets supplemented with ZnSO₄ or Zn-Met at 30mg/kg. Initial bodyweight and final body weight, serum and liver zinc contents, activity of serum AKP, plasmagrowth hormone concentration and the levels of GHR and IGF-I mRNA were determined.The results showed that, weight gain of the mice was enhanced by both forms of zinc, Zn-Metwas more efficient than ZnSO₄ （P<0.05） at the early growth stage. Serum, liver zinc contentsand serum alkaline phosphtase （AKP） activity were increased by both forms of zinc, thosetreated with Zn-Met were somewhat higher than with ZnSO₄ （P>0.05）. The both forms ofzinc had no effect on GH concentration and the expression of GHR mRNA （P>0.05）, whilethey markedly （P<0.05） up-regulated the expression of IGF-I mRNA. As compared to ZnSO₄,Zn-Met significantly increased the weight gain and the abundance of IGF-I mRNA （P<0.05）.3. Transport and regulation mechanism of zinc sulfate and zinc methionine in miceNinety male KM mice were used to investigate the metabolism regulation of ZnSO4 andZn-Met in the intestine of mice. ZnT1 （zinc transporter）, ZnT4, DCT1 （divalent cationtransporter） and MT-1 （metallothionein） mRNAs were determined using semi-quantificationreverse transcript polymerase chain reaction （RT-PCR）. The results showed that ZnSO4 andZn-Met decreased the expression of ZnT1, ZnT4 and DCT1 mRNAs （P<0.05）. Theexpression of ZnT1 and DCT1 mRNAs were down-regulated significantly by Zn-Met ascompared to ZnSO4 （P<0.05）;ZnT4 mRNA abundance showed no difference between thetwo zinc sources （P>0.05）. Both ZnSO4 and Zn-Met significantly increased MT-1 mRNA inliver and intestine （P<0.05）. The expressions of DCT1 mRNA were down-regulated whileMT-1 mRNA expression was up-regulated more efficiently by Zn-Met as compared to ZnSO4（P<0.05）. According to above results, the two forms of zinc might be transported or regulatedby different ways.4. Effect of zinc sulfate and zinc methionine on immunity of miceOne hundred and twenty weanling mice （KM） were allotted at random into 3 treatments,some immunology indexes were determined to investigate the effect of the two forms of zincon immunity of weanling mice. The results showed that zinc supplementation in basal dietenhanced immune response;indexes of thymus, ANAE+, IL-2, SI, IgA, IgG, and IgM were allincreased with supplementation of both forms of zinc （P<0.05）;index of spleen, hemolysin,IL-1 in zinc treatment of ZnSO4 showed no difference （P>0.05） with the control, while intreatment of Zn-Met, it showed markedly difference （P<0.05）;There were no significantdifference of indexes of liver and kidney, serum protein and albumin contents among the theretreatments （P>0.05）. Based on all indexes except indexes of kidney and liver, SI and IgM, theimmunity was improved more efficiently by Zn-Met than ZnSO4.5. Effect of different zinc sources on the ability of antioxidation of miceThis experiment was conducted to investigate the effect of zinc sulfate and zinc methionineon the redox of weanling mice （KM）. One hundred and twenty weanling mice （KM） wereallotted at random into 3 treatments. Nitric oxide synthase （NOS）, glutathione peroxidase（GSH-Px）, total superoxide dismutase （T-SOD） and CuZn-SOD, the content of nitric oxide（NO） and total antioxidation capacity （T-AOC） were determined. The result indicated that,oxidation damage was induced by zinc deficiency based on the increased plasma MDAconcentration;the activity of GSH-Px was enhanced significantly with zinc supplementation（P<0.05）;while the two zinc sources showed no difference. T-AOC, T-SOD and CuZn-SODwere increased by zinc supplementation （P<0.05）, and zinc methionine exerted effectivelythan zinc sulfate （P<0.05）. The content of NO was decreased with zinc supplementation（P<0.05）, Zn-Met did more effectively than ZnSO4 （P<0.05）. The activity of NOS wasincrease slightly by zinc supplementation （P>0.05）. Based on above results, it was concludedthat Zn-Met improved the ability of antioxidation more effectively than ZnSO4.6. Effects and mechanism of zinc sources and levels on apoptosis of thymocytes culturedin vitroThis experiment was conducted to investigate the effects of different zinc sources andlevels on apoptosis and their mechanism. Dexamethasone was used to make the apoptosismodel of thymocytes;zinc sulfate and zinc methionine were added to the medium with thelevels of 0, 50, 100, 500, 1000μM. The activities of AKP, GSH-Px, CuZn-SOD andintracellular calcium concentration, the percentage of apoptosis nuclei were determined. Theresults showed that, both ZnSO4 and Zn-Met increased the activities of cells which weredrceased by dexamethasone;Zn-Met was more efficient than ZnSO4. Both forms of zincinhibited apoptosis, the modulation relied on the dose of zinc. With 50, 100, 1000μM, theability of Zn-Met inhibiting apoptosis was less efficient than ZnSO4 （P<0.05）, and theyshowed no difference on modulating apoptosis with 500μM （P>0.05）. Intracellular calciumconcentrations of cells cultured with Zn-Met were higher than those cultured with ZnSO4 ofthe same level. Zinc supplementation decreased the concentration of intracellular calciumsignificantly （P<0.05）, but increased the activities of GSH-Px and CuZn-SOD （P<0.05） inboth the extract and the supernatant of the cells cultured, the AKP activity in the supernatantof the culture fluids was also increased by zinc supplementation （P>0.05）. Zn-Met did moreefficiently than ZnSO4 in improving the ability of antioxdiation of the cells. It was concludedthat both forms of zinc inhibited apoptosis of thymocytes induced by glucocorticoid;themechanism involved the exchange of intracellular calcium and the redox of cells;and the twoforms of zinc inhibited apoptosis by different ways.7. The regulation mechanism of different zinc sources on apoptosisDexamethasone was used to make the apoptosis model of thymocytes. ZnSO4 and Zn-Metwas added to the medium at 1000μM, the percentage of apoptosis nuclei were determined byflow cytometric analysis, bcl-2, bax and caspase-3 mRNAs abundance were measured byRT-PCR. The results showed that both ZnSO4 and Zn-Met inhibited apoptosis induced bydexamethasone （P<0.05）;no difference was shown between the two forms of zinc （P>0.05）.Both forms of zinc decreased the up-regulation of bcl-2, bax and caspase-3 mRNAsexpression by dexamethasone （P<0.05）. Cell availability and caspase-3 mRNA expression ofcells cultured with Zn-Met were higher than those cultured with ZnSO4 （P<0.05）. Thepercentage of apoptosis nuclei, bcl-2 and bax mRNAs expression were similar within the twotreatments （P>0.05）. It was concluded that both forms of zinc could inhibit apoptosis ofthymocytes induced by glucocorticoid;the mechanism involved the regulating ability of zincon genes expression, and the two forms of zinc regulated apoptosis by different ways.From above results, it was concluded that, both ZnSO4 and Zn-Met improved the growth ofanimals by up-regulating the expression of IGF-I mRNA. Zn-Met enhanced the weight gain,up-regulated IGF-I mRNA expression and was transported more efficiently than ZnSO4.DDRT-PCR was an effective way to identify unknown genes regulated by different zincsources. Both forms of zinc improved the immunity and ability of antioxidation in mice, whileZn-Met showed more effectively than ZnSO4. In inhibiting apoptosis, Zn-Met was moreefficiently in increasing the ability of antioxidation and the ratio of bcl-2 and bax mRNAs,and less efficient in decreasing apoptosis happening, the increment of intracellular calciumand caspase-3 mRNA expression. In addition, Zn-Met was less harmful to cells cultured.更多还原