【作者】 孙国君；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 本研究以断奶仔猪和小肠上皮细胞（IPEC-J2）为试验模型,通过四个试验考察了锌对不同免疫状态下仔猪和小肠上皮细胞免疫功能的调节作用。试验一日粮锌水平对仔猪生长性能和免疫功能的影响本试验采用单因子设计,选用72头28日龄断奶的杂交仔猪（DLY）,在基础日粮中分别添加0、60、120mg/kg锌（ZnSO₄）,研究了日粮锌水平对断奶仔猪生长性能、组织锌含量、碱性磷酸酶活性、外周血淋巴细胞转化率、牛血清白蛋白（BSA）抗体水平、肝脏金属硫蛋白（MT）含量及MTmRNA水平的影响。试验全期共21天。试验结果表明:在本试验条件下,不同锌添加水平对断奶仔猪生长性能、组织锌含量、外周血淋巴细胞转化率、肝脏MT含量及MTmRNA水平无显著影响（P＞0.05）;随日粮锌水平增加,仔猪血清碱性磷酸酶（AKP）活性极显著增加（P＜0.01）,牛血清白蛋白（BSA）抗体水平在免疫后第7天,随日粮锌水平增加而显著降低（P＜0.05）,在第14天则不受影响。研究结果证明,NRC（1998）推荐的锌水平可满足断奶仔猪最佳生长速度和免疫应答的需要。试验二日粮锌水平和免疫应激对仔猪生长性能和免疫功能的影响本试验采用2×3因子设计,（1）免疫应激处理（注射大肠杆菌脂多糖或生理盐水）,（2）日粮类型（基础日粮中添加0、60、120mg/kg锌）。在试验的第7天,每日粮处理组随机选取半数仔猪按200μg/kg体重的剂量腹腔注射脂多糖（LPS）,另一半注射同样剂量的生理盐水。试验全期共21天。注射LPS后3h,每个重复选择1头猪采集血清,测定细胞因子白介素1（IL-1）和白介素2（IL-2）浓度。注射LPS后第2天,每个重复1头猪从前腔静脉采血测定外周血淋巴细胞转化率;另外选1头猪从耳后肌肉注射牛血清白蛋白（BSA）,并于注射后第7和12天从前腔静脉采血,测定血清BSA抗体水平。试验结果表明:LPS刺激显著降低了仔猪注射后第一周的日增重和采食量（p＜0.01）,但对饲料转化效率无影响。LPS刺激提高了外周血淋巴细胞转化率（p＜0.05）和血清IL-1浓度（p＜0.01）,但未影响血清IL-2浓度和BSA抗体水平。在LPS刺激后第一周,随日粮锌水平增加,仔猪日增重有增加的趋势（p＜0.10）,而采食量和饲料转化效率未受影响。随着锌水平提高,淋巴细胞转化率有增加趋势（p＜0.10）。日粮处理没有影响BSA抗体水平、血清IL-1、IL-2浓度。提示经历短期免疫应激的仔猪对锌的需要量要高于健康仔猪。试验三锌和脂多糖对小肠上皮细胞（IPEC-J2）免疫功能的影响肠上皮细胞在肠腔抵御病原体和细菌的先天性免疫应答中,起到了非常重要的作用。在本次试验中,我们以肠细胞因子IL-8、TNF-α和TGF-β的基因表达为反应标识,考察了锌和脂多糖（LPS）对猪小肠上皮细胞系（IPEC-J2）免疫功能的影响。试验结果表明:1.IPEC-J2细胞内锌含量随培养基锌浓度升高而增高。与普通培养基对照组（6μmol/L锌）相比较,50μmol/L和100μmol/L硫酸锌组细胞锌含量分别提高55.69%和98.75%,差异极显著（P＜0.01）;50μmol/L和100μmol/L乙酸锌组细胞锌含量分别提高67.65%和116.58%（P＜0.01）。IPEC-J2细胞的ZnT1和MTmRNA表达随着培养基锌浓度升高而增高,与对照组相比差异均显著（P＜0.05）;在培养基锌浓度为50μmol/L时,DMT1mRNA表达量达到峰值,显著高于对照组（P＜0.01）,达到100μmol/L时,与培养基锌浓度50μmol/L相比较。DMT1mRNA表达水平有所下降,但仍高于对照组（P＜0.05）。2.在脂多糖刺激下,与对照组相比较,细胞锌含量下降5.10%,而培养基锌浓度为50μmol/L和100μmol/L的硫酸锌组细胞锌含量分别提高27.36%和45.64%（P＜0.01）;培养基锌浓度为50μmol/L和100μmol/L的乙酸锌组细胞锌含量分别提高26.11%和73.07%（P＜0.01）。LPS刺激对小肠上皮细胞ZnT1、DMT1和MTmRNA表达量均无显著影响（P＞0.05）。IPEC-J2细胞经内毒素刺激后,随培养液中锌浓度的增加,ZnT1mRNA（P＜0.01）和MTmRNA（P＜0.05）的表达显著提高。在培养基锌浓度为50μmol/L时,DMT1mRNA表达量达到最高峰,显著高于对照组（P＜0.01）,锌浓度为100μmol/L时,与培养基锌浓度50μmol/L相比较,DMT1mRNA表达水平显著下降（P＜0.05）,与对照组DMT1mRNA表达量相同。3.培养液中不同锌源和锌水平没有影响细胞IL-8、TNF-α和TGF-β的基因表达水平。4.LPS刺激可使IL-8（P＜0.01）和TNF-α（P＜0.05）的mRNA表达水平显著上调,TGF-β的mRNA表达水平显著下调（P＜0.01）;且同对照组相比较,随着锌浓度的提高,IL-8、TNF-α和TGF-β基因表达量的增加（P＜0.05）。试验四锌和环磷酰胺对小肠上皮细胞（IPEC-J2）免疫功能的影响环磷酰胺是一种常用抗肿瘤药物,是常用免疫抑制剂。现已证明环磷酰胺通过降低抗体产量抑制B淋巴细胞的免疫功能,另外环磷酰胺也可促进TNF-α诱导的凋亡细胞死亡。为了探索锌和环磷酰胺对猪小肠上皮细胞免疫功能的影响,在本次试验中,我们以肠细胞因子IL-8、TNF-α和TGF-β的基因表达位反应标识,测定了锌和脂多糖（LPS）对猪小肠上皮细胞系（IPEC-J2）免疫功能的影响。试验结果表明:1.无论有无环磷酰胺刺激,IPEC-J2细胞内锌含量均随培养基锌浓度升高而增高。2.在环磷酰胺作用下,细胞锌含量下降7.07%,而培养基锌浓度为50μmol/L和100μmol/L硫酸锌组细胞锌含量分别提高4.54%和39.91%（P＜0.01）;培养基锌浓度为50μmol/L和100μmol/L乙酸锌组细胞锌含量分别提高7.92%和22.52%（P＜0.01）。与对照组比较,ZnT1mRNA表达量显著增加（P＜0.01）,而DMT1和MTmRNA的表达量及显著降低（P＜0.01）。随着锌水平的提高,ZnT1和MTmRNA表达量显著提高（P＜0.01）。DMT1mRNA的表达量显著下降（P＜0.05）。3.环磷酰胺刺激可使IL-8和TNF-α的基因表达量被显著下调（P＜0.05）,而对TGF-β的基因表达量无影响。与对照组（环磷酰胺组）比较,硫酸锌水平提高,使IL-8mRNA的表达量显著升高（P＜0.01）,而对TGF-β和TNF-α的基因表达量则影响不大,但随锌水平的增加,两者的mRNA表达水平有上调趋势;乙酸锌水平提高,使IL-8和TNF-α的基因表达量显著增加（P＜0.05）。上述结果揭示,NRC（1998）推荐的锌水平可满足断奶仔猪最佳生长速度和免疫应答的需要。提示经历短期免疫应激的仔猪对锌的需要量要高于健康仔猪。锌对处于免疫应激状态下的小肠上皮细胞的免疫功能有调节作用。更多还原

【Abstract】 Four in vivo and in vitro experiments were conducted in tiffs study to investigate nutritional regulatory effects of zinc on immune response in weanling pigs and porcine epithelial cells.Exp.1 Effects of dietary zinc level on performance and immune response of weanling pigsAn experiment was conducted to determine the effect of dietary zinc level on growth performance and immune function in normal weanling pigs.Treatments consisted of the following:1) a corn-soybean meal basal diet contained 36.75 mg/kg total Zn,2) basal diet+60 mg/kg added Zn as ZnSO₄,3) basal diet+120mg/kg added Zn as ZnSO₄.Each diet was fed to six pens of four pigs per pen.In Exp 1,the dietary zinc level had no effect on average daily growth（ADG）,average daily feed intake（ADFI）,or Feed conversion ratio（FCR）.Concentrations of tissue and serum zinc were not affected.AIK ativity was increased.Metalodlionein（MT） concentration,MTmRNA level were not affected.Peripheral blood lymphocyte proliferation（PBLP） was not affected by dietary treatments.Supplementation of 120 mg/kg Zn decreased（p＜0.05） the antibody response to bovine serum albumin（BSA） on d 7 compared with pigs fed the basal diet,but not on d 14.Results indicate that the level of Zn recommended by NRC（1998） for weanling pigs was sufficient for optimal growth performance and immune responses.Exp.2 Effects of dietary zinc level and an inflammatory challenge on performance and immune response of weanling pigsAn experiments were conducted to determine the effect of dietary zinc level on growth performance and immune function in immunologically challenged weanling pigs.Treatments consisted of the following:1) a corn-soybean meal basal diet contained 36.75 mg/kg total Zn,2) basal diet+60 mg/kg added Zn as ZnSO₄,3) basal diet+120mg/kg added Zn as ZnSO₄.Each diet was fed to six pens of three pigs per pen.In Exp 2,LPS challenge did not effect on ADG,ADFI and FCR in the entire trial（from d 0 to 21）.LPS challenge significantly decreased ADG and ADFI （p＜0.01） from d 7 to 14,but FCR was not affected.LPS challenge increased PBLP （p＜0.05） and serum concentration of interleukin-1（IL-1）（p＜0.01）,whereas the antibody response to BSA and serum concentration of interleukin-2（IL-2） were not affected.Supplementation of Zn did not affect ADFI and FCR from d 7 to 14,but there was a trend for ADG to be enhanced with Zn supplementation（p＜0.10）. Supplementation of Zn tended to increase PBLP（p＜0.10）.Dietary treatment had no effect on the antibody response to BSA,concentrations of serum IL-1 and IL-2. Results indicate that the level of Zn recommended by NRC（1998） for weanling pigs was sufficient for optimal growth performance and immune responses.Zn requirements may be higher for pigs experiencing an acute phase response than for healthy pigs.Exp.3 Effects of zinc level and LPS on immune response of swine intestinal epithelial cell lines（IPEC-J2）Intestinal epithelial cells play an important role in the innate immune response against pathogenic bacteria in the lumen of the gut.In the present study,we determined the effects of LPS and zinc on immune response of porcine epithelial cell lines（IPEC-J2） by regarding mRNA expression of proinflammatory mediators,such as cytokines（TNF-α,TGF-β） and chemokine（IL-8） as representative marker.The results were as follows:1.The zinc content of cells was enhanced with Zn supplementation.Treatments with 50μmol/LZnSO₄ and 100μmol/LZnSO₄ increased cellular zinc uptake by 55.69%and by 98.75%,respectly,compared with control cells.Treatments with 50μmol/L and 100μmol/L zinc acetate increased zinc content of cells by 67.65% and 116.58%（P＜0.01）,respectively.The expression of ZnT1,DMT1 and MT were upregulated with Zn concentration of medium（P＜0.05）.In cells treated with 50μmol/L,the expression of DMT1mRNA reacheded peak（P＜0.01）.2.After LPS challenge,zinc content of cells was decreased by 5.10%.However, treatments with 50μmol/LZnSO₄ and 100μmol/LZnSO₄ increased zinc content of cells by 27.36%and by 45.64%（P＜0.01） 98.75%,respectly,compared with control cells.Treatments with 50μmol/L and 100μmol/L zinc acetate increased zinc content of cells by 26.11%（P＜0.05） and 73.07%（P＜0.01）,respectively.LPS challenge had no feffect on the expression of IL-8,TNF-αand MT（P＞0.05）.The expression of ZnT1mRNA（P＜0.01） and MTmRNA（P＜0.05） were increased with Zn supplementation.In cells treated with 50μmol/L,the expression of DMT1mRNA reacheded peak（P＜0.01）. 3.zinc resources and zinc levels had no effect on the expression of of TNF-α,IL-8 and TGF-β.4.LPS challenge upregulated the expression of IL-8（P＜0.01） and TNF-α（P＜0.05）,but downregulated the expression of TGF-β（P＜0.01）.After LPS challenge,supplementation of Zn upregulated the expression of IL-8,TNF-αand TGF-β,compared with untreated cells.Exp.4 Effects of zinc level and Cyclophosphamide（CP） on immune response of swine intestinal epithelial cell lines（IPEC-J2）Cyclophosphamide（CP） is a widely used anti-cancer prodrug,is effective for autoimmune diseases.It has been shown that CP suppresses B-cell function with decreased antibody production and enhances TNF-α-induced apoptotie cell death. In this study,We determined the effects of Cyclophosphamide（CP） and zinc on immune response of porcine epithelial cell lines（IPEC-J） by regarding mRNA expression of proinflammatory mediators,such as cytokines（TNF-a,TGF-β） and chemokine（IL-8） as representative marker.The results were as follows:1.The zinc content of cells was enhanced with Zn supplementation.2.After Cyclophosphamide challenge,zinc content of cells was decreased by 7.07%. However,treatments with 50μmol/LZnSO₄ and 100μmol/LZnSO₄ increased zinc content of cells by 4.54%and by 39.91%,respectly,compared with control cells. Treatments with 50μmol/L and 100μmol/L zinc acetate increased cellular zinc uptake by 7.92%and 22.52%,respectively.The expression of ZnT1mRNA was upregulated by（P＜0.01）.However,the expression of DMT1 and MT were downregulated by （P＜0.01）,compared with control cells.The expression of ZnT1mRNA and MTmRNA were upregulated by（P＜0.01） with Zn supplementation.But,the expression of DMT1mRNA was downregulated by（P＜0.05）3.After Cyclophosphamide challenge,the expression of IL-8 and TNF-αwere downregulated by（P＜0.05）,but the expression of TGF-βwas affected.Compared with control cells,the expression of IL-8 mRNA was upregulated（P＜0.01）.However, the expression of TGF-βand TNF-αwere not affected.Zn supplementation upregulated the expression of TGF-βand TNF-α（P＞0.05）.Zn（zinc acetate）supplementation upregulated the expression of TGF-β,IL-8 and TNF-α（P＜0.05）.zinc resources and zinc levels had no effect on the expression of TNF-α, IL-8 and TGF-β.ImplicationsThese results in vitro and in vivo showed that the level of Zn recommended by NRC（1998） for weanling pigs was sufficient for optimal growth performance and immune responses.Zn requirements may be higher for pigs experiencing an acute phase response than for healthy pigs.Zinc play an important role in the regulatory of immune respone in immunologically challenged weanling pigs and porcine epithelial cells更多还原