【作者】 蒋守群；

【导师】 吴天星； 蒋宗勇；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 本研究用大豆异黄酮SI处理离体培养岭南黄羽肉鸡骨骼肌细胞,研究大豆异黄酮SI对骨骼肌细胞的增殖和抗氧化作用的影响;研究金雀异黄酮(GEN)对骨骼肌细胞脂质过氧化、抗氧化酶活性和基因表达的影响；用H2O2/FeSO4氧化岭南黄羽肉鸡骨骼肌细胞造成氧化模型,研究在氧化环境中GEN和大豆异黄酮SI的抗氧化作用、对细胞膜流动性的影响及其对抗氧化酶基因表达的影响；在42日龄岭南黄羽肉公鸡饲粮中添加不同浓度梯度的大豆异黄酮SI,研究大豆异黄酮SI对岭南黄羽肉公鸡生长性能、胴体指标、肉品质特性、血浆、肝脏和胸肌中生化指标的影响以及胸肌中抗氧化酶基因表达的影响；采用2(大豆异黄酮SI添加)×2(鱼油氧化处理)因子随机分组设计,在42日龄岭南黄羽肉公鸡新鲜鱼油或氧化鱼油饲粮中添加或不添加大豆异黄酮SI,研究在两种鱼油饲粮中添加大豆异黄酮SI对岭南黄羽肉公鸡生长性能、胴体指标、肉品质特性、血浆、肝脏和胸肌中生化指标的影响以及胸肌中抗氧化酶基因表达的影响；旨在探讨大豆异黄酮对岭南黄羽肉鸡的影响及其抗氧化作用机理,为大豆异黄酮作为一种抗氧化饲料添加剂在肉鸡生产中的应用提供理论依据。试验一：选用20日龄岭南黄羽肉鸡鸡胚在无菌环境下分离腿部骨骼肌,经过剪碎、胶原酶Ⅰ和胰蛋白酶消化,通过100目和200目筛过滤,滤液转移到10ml离心管中1700rpm离心、清洗得到骨骼肌原代细胞,用计数板计数,根据计数结果将细胞液稀释至5.0×105/ml,然后接种至培养瓶中,用完全培养基在二氧化碳培养箱培养48小时,再经0.25%胰蛋白酶消化传代,采用差速贴壁法得到骨骼肌卫星细胞,以5.0×105/ml浓度接种至96孔板和24孔板,试验分5组,各组无血清培养液中分别添加0、12.5、25、50、75和100μmol/L的大豆异黄酮SI(所用大豆异黄酮为广东省农业科学院畜牧研究所研制产品,纯度为98.5%以上),大豆异黄酮预先溶解于二甲基亚砜中,并保持各组中二甲基亚砜浓度一致,所有培养液中均添加80μmol/L H2O2/FeSO4进行氧化处理建立氧化模型,每组6个重复(孔),研究了大豆异黄酮对肉鸡骨骼肌卫星细胞增殖和抗氧化作用的影响,试验观察了骨骼肌卫星细胞经大豆异黄酮处理后的形态变化和采用MTT法测定骨骼肌卫星细胞的增殖情况,结果表明,在氧化环境中,大豆异黄酮可减少骨骼肌卫星细胞死亡,而且添加大豆异黄酮均促进骨骼肌卫星细胞的增殖(p<0.01),各大豆异黄酮处理组骨骼肌卫星细胞经MTT染色和SDS处理后的吸光度分别较对照组提高45.90%(P<0.01)、58.64%(P<0.01)、60.21%(P<0.01)、46.42%(P<0.01)和25.48%(P<0.05)。添加25,50,75和100μmol/L SI使细胞培养上清液中T-SOD活性分别提高17.02%(P<0.01),13.00%(P<0.05),13.26%(P<0.05)和11.89%(P<0.05),添加25μmol/L SI提高GSHPx活性90.68%(p<0.05),75和100μmol/L SI处理组CAT活性分别比对照组提高49.17%(P<0.01)和49.13%(P<0.05),SI有提高细胞上清液中T-AOC的趋势(P>0.05),添加75和100μmol/L大豆异黄酮处理显著降低细胞CK的分泌(P<0.05)。总之,大豆异黄酮SI能促进骨骼肌卫星细胞在氧化环境中的增殖,提高骨骼肌卫星细胞的抗氧化能力。试验二：为研究大豆异黄酮主要成分金雀异黄酮(GEN)对离体培养的鸡骨骼肌细胞脂质过氧化、抗氧化酶活性和基因表达的影响,岭南黄羽肉鸡骨骼肌细胞接受不同浓度的GEN处理48h后,测定细胞培养上清液中谷胱甘肽过氧化物酶、超氧化物歧化酶和CAT活性以及丙二醛含量,应用TaqMan实时荧光定量PCR法测定GEN对骨骼肌细胞中谷胱甘肽过氧化物酶基因表达的影响。结果表明,与空白对照组相比,添加GEN组骨骼肌细胞培养上清液中MDA含量显著降低(p<0.05),添加1μmol/L和16μmol/L GEN显著提高了鸡骨骼肌细胞培养上清液中SOD活性(p<0.05),添加4μmol/L和16μmol/L GEN显著提高了GSHPx活性(p<0.05),细胞培养液中添加GEN具有提高GSHPx mRNA拷贝数趋势,但各组间无显著差异(p>0.05)。由此可见,添加金雀异黄酮能抑制鸡骨骼肌细胞的脂质过氧化,增强鸡骨骼肌细胞的抗氧化能力。试验三：岭南黄羽肉鸡骨骼肌细胞以5.0×105/ml浓度接种至6孔板和24孔板,试验分10组,组1为正常空白对照组,未添加H2O2/FeSO4和大豆异黄酮,组2为氧化细胞组,仅添加H2O2/FeSO4但未添加大豆异黄酮,组3、4、5、6在均添加50μmol/L H2O2/FeSO4基础上分别10、20、40、80μmol/L的GEN,组7、8、9、10在均添加50μmol/L H2O2/FeSO4基础上分别10、20、40、80μmol/L的SI, GEN和SI预先溶解于二甲基亚砜中,并保持各组中二甲基亚砜浓度一致,每组6个重复(孔),研究了GEN和SI对岭南黄羽肉鸡骨骼肌细胞抗氧化状况、细胞膜流动性和骨骼肌细胞中CAT和GSHPx mRNA表达量的影响。结果表明,H2O2/FeSO4氧化组细胞培养上清液MDA含量略高于其它各组(p<0.1), T-SOD活性最低,极显著低于其它各组(p<0.01), GSHPx活性也最低,极显著低于添加20、40、80μmol/L GEN组和添加20、80μmol/L SI组(p<0.01)。添加40、80μmol/LSI组骨骼肌细胞培养上清液CK活性极显著低于除80μmol/LGEN组的其它各组(p<0.01),添加80μmol/LGEN组CK活性显著低于空白对照组和H2O2/FeSO4氧化组(p<0.05)。H2O2/FeSO4氧化组LDH活性极显著高于空白对照组、40μmol/L GEN组和添加SI的各组(p<0.01)。岭南黄羽肉鸡骨骼肌细胞经H2O2/FeSO4处理后,细胞膜荧光偏振值p和微粘度η极显著增加(P<0.01)。添加80μmol/L GEN或者SI组细胞膜p值极显著低于H2O2/FeSO4氧化处理组(P<0.01),但与正常未氧化组细胞无显著差异(p>0.05)。与H2O2/FeSO4氧化处理组比较,添加各水平GEN或者SI均极显著降低了细胞膜η值(P<0.01),下降幅度以80μmol/L浓度组最大,分别为87.53%和89.51%。各异黄酮水平和种类之间无显著差异(p>0.05)。添加20μmol/L SI或者20μmol/LGEN组的GSHPx基因表达水平极显’著高于氧化细胞组(P<0.01),提示大豆异黄酮SI和GEN具有促使离体培养鸡骨骼肌细胞的GSHPx基因表达的作用。总体上,GEN和SI能够从分子水平调控抗氧化酶活性,提高骨骼肌细胞在氧化环境中的抗氧化能力,改善细胞膜流动性,抵抗氧化损伤,SI的抗氧化作用效果较GEN更强。试验四：选用42日龄体重约1.03kg健康、发育良好的岭南快大黄羽肉鸡公鸡,根据体重随机分为5个处理,每处理均6个重复(栏),每重复50只鸡,试验采用玉米-大豆浓缩蛋白型基础饲粮,各处理饲粮中分别添加0、10、20、40、80mg/kg大豆异黄酮SI,其他营养成分一致,试验鸡自由采食粉料、自由饮水,地面平养。试验期21日。研究不同浓度SI对黄羽肉鸡生产性能、肉品质和机体抗氧化性能的影响。试验结果表明：在饲粮中添加10、20mg/kgSI分别提高试鸡平均日增重13.63%(p<0.01)和16.19%(p<0.01),添加20mg/kgSI组试鸡平均日采食量较对照组提高11.20%(p<0.01),添加10mg/kg SI显著降低试鸡料重比5.53%(p<0.05)。在肉品质方面,添加40 mg/kg SI显著增加试鸡胸肌肌纤维密度(p<0.05),添加80 mg/kg大豆异黄酮极显著增加试鸡胸肌肌纤维密度(p<0.01)。添加40mg/kg SI组试鸡宰后24h胸肌肉色a*值略高于空白对照组(p<0.1),试鸡宰后45min和96h胸肌肉色L*值较空白对照组分别降低3.89%(p<0.05)和3.81%(p<0.05),试鸡宰后45mmin胸肌系水力提高17.24%(p<0.1),添加各水平SI均极显著提高了试鸡宰后24h和72h胸肌系水力(p<0.01),添加10mg/kg、20mg/kg和40mg/kg SI也极显著提高了试鸡宰后96h胸肌系水力(p<0.01),添加80mg/kg SI组试鸡宰后45min胸肌pH值提高(p<0.1),添加20mg／kg和40mg/kg SI极显著提高了试鸡宰后24h胸肌pH值(p<0.01),添加40mg/kgSI极显著提高了试鸡宰后96h胸肌pH值(p<0.01),试鸡宰后胸肌中LD含量随着贮藏时间延长而呈逐渐增加趋势,添加10 mg/kg、20 mg/kg和40 mg/kg SI使试鸡宰后45min胸肌中LD含量分别降低17.29%(p<0.05)、13.16%(p<0.05)和16.92%(p<0.05),添加40 mg/kg和80 mg/kg SI使试鸡血浆中总抗氧化能力分别提高109.50%(p<0.01)和129.59%(p<0.01),使T-SOD活性分别提高7.60%(p<0.1)和5.86%(p<0.1),添加80 mg/kg SI组试鸡血浆中CAT活性提高37.06%(p<0.1),添加40 mg/kg SI组试鸡胸肌中CAT活性提高70.61%(p<0.05),添加各水平SI均显著降低试鸡肝脏中MDA含量(p<0.05),添力10 mg/kg和20 mg/kg SI显著降低了试鸡血浆中MDA含量(p<0.05)。试鸡宰后胸肌中MDA含量随着贮藏时间延长而呈逐渐增加趋势,在宰后45min测得,添加20 mg/kg、40 mg/kg和80 mg/kg SI均显著或极显著降低了胸肌中MDA含量(p<0.05或p<0.01),添加20 mg/kg、40 mg/kg和80 mg/kg SI显著降低了试鸡宰后72h胸肌中MDA含量(p<0.05),添加10mg/kg SI组试鸡宰后45mmin胸肌中T-SOD活性显著降低(p<0.05),添加20 mg/kg、40 mg/kg和80 mg/kg大豆异黄酮SI使宰后45mmin胸肌中T-SOD活性极显著降低(p<0.01),宰后12h测得的结果显示,添加40 mg/kg SI显著提高了胸肌中T-SOD活性(p<0.05),添加80 mg/kg SI组试鸡胸肌中T-SOD活性极显著高于对照组(p<0.01)和其它SI水平组(p<0.01),宰后48h测得,添加20 mg/kg、40 mg/kg和80 mg/kg SI组试鸡胸肌中T-SOD活性显著高于对照组(p<0.05),添加20 mg/kg和40 mg/kg SI极显著提高了试鸡宰后72h胸肌中T-SOD活性(p<0.01),添加SI均显著或极显著提高岭南黄肉鸡胸肌中GSHPx mRNA表达量(p<0.05或p<0.01)。上述结果提示：添加大豆异黄酮SI增强肉鸡抗氧化能力,改善生长性能和新鲜肉与肉品贮藏过程中的肉品质,对延长鸡肉的货架期十分有利,综合考虑生产性能、肉品质和各项生化指标得出,43-63日龄阶段岭南黄羽肉鸡饲粮中添加20mg/kg大豆异黄酮SI较适宜。试验五：选用42日龄健康、发育良好的岭南快大黄羽肉鸡公雏作为试验鸡,根据体重随机分为4个处理,每处理6个重复,每重复40只鸡,试验采用2(油脂氧化)×2(大豆异黄酮添加)因子随机分组设计,组1试鸡饲粮用新鲜鱼油未添加大豆异黄酮SI,组2为新鲜鱼油+大豆异黄酮SI组,组3试鸡饲粮用氧化鱼油未添加大豆异黄酮SI,组4为氧化鱼油+大豆异黄酮SI组,研究了在氧化鱼油和新鲜鱼油饲粮中添加SI对岭南黄肉公鸡生产性能、胴体品质、肉品质和抗氧化作用机制的影响。试验结果表明：SI处理和鱼油氧化处理对试鸡生产性能无显著影响,鱼油氧化处理极显著增加胸肌肉色L*P<0.01),极显著降低b*值(p<0.01),显著降低胸肌肉色a*值(p<0.05),鱼油氧化处理极显著影响试鸡血浆中T-AOC和CK活性(p<0.01)。SI处理显著影响试鸡宰后45min胸肌中MDA含量(p<0.05),极显著影响宰后96h胸肌中MDA含量(p<0.01)；鱼油氧化处理对宰后24h和96h胸肌中MDA含量均产生极显著影响(p<0.01)；SIx鱼油氧化处理对宰后96h胸肌中MDA含量产生显著互作效应(p<0.05)。SI处理极显著降低试鸡宰后45min胸肌中LD含量(p<0.01);SIx鱼油氧化处理对宰后96h胸肌中LD含量产生极显著互作效应(p<0.01)。SI处理极显著提高试鸡宰后胸肌中CAT和T-SOD活性(p<0.01),显著提高T-AOC和GSHPx活性(p<0.05)；鱼油氧化处理显著降低T-AOC(p<0.05);SIx鱼油氧化处理对宰后CAT活性和T-AOC影响产生显著互作效应(p<0.05),对GSHPx活性具有极显著互作效应(p<0.01)。SI处理对胸肌中GSHPx mRNA表达量具有显著影响(p=0.029)。上述结果提示：鱼油氧化处理对鸡肉品质和抗氧化状况产生不利影响,在鱼油饲粮中补充大豆异黄酮SI提高了肉鸡抗氧化状况和肌肉的抗氧化稳定性,抵抗脂质过氧化,保护肉鸡胸肌肉免受氧化损失,补充大豆异黄酮SI能从分子水平促进胸肌中GSHPx mRNA的表达,间接发挥抗氧化效应。以上5个试验结果表明,大豆异黄酮能够促进岭南黄羽肉鸡骨骼肌细胞增殖,增强抗氧化能力,抑制氧化损失；大豆异黄酮能够改善岭南黄羽肉鸡生长性能和肉品质和抗氧化状况。其作用机制可能为：大豆异黄酮能抵抗脂质过氧化,促进抗氧化酶基因的高丰度表达,提高抗氧化酶活性,改善细胞膜的流动性,保护细胞膜。更多还原

【Abstract】 Five studies were designed to investigate the effects of soy isoflavones on antioxidant property in chick skeletal muscle cells (SMC) and Lingnan yellow male broilers. The first three studies were carried out to investigate the protective effects of a synthetic isoflavone(SI) and genistein(GEN) on antioxidative defense system and membrane fluidity in chick skeletal muscle cells in vitro. The last two studies were conducted to investigate the effects of a synthetic isoflavone on growth performance, meat quality, and antioxidative property in Lingnan yellow male broilers.In study 1, the antioxidant and protective properties of SI were investigated using chick SMC (leg) isolated from 20-day-old Lingnan yellow broiler chick embryo. Chick SMC were cultured in Dulbecco’s modified Eagle’s medium added with 0,12.5,25,50, 75 and 100μmol/L SI respectively under 80μmol/L H2O2/FeSO4 conditions. After 48h incubation, SI reduced the loss of SMC under oxidative stress by H2O2/FeSO4 The addition of SI significantly promoted SMC proliferation (p<0.01). Supplementation with 25,50,75 and 100μmol/L SI enhanced the activity of total superoxide dismutase (T-SOD) by 17.0%,13.0%,13.3% and 11.9%respectively in the supernatant of media (p<0.05). Compared to the control, the activity of glutathione peroxidase (GSHPx) increased by 90.7% at 25μmol/L concentration of SI (p<0.05). The activity of catalase (CAT) increased by 49.2%and 49.1%respectively at 75 and 100μmol/L SI (p<0.01). The concentration of creatine kinase (CK) in the media decreased by 61.6%and 60.6% respectively at 75 and 100μmol/L SI (p<0.01). Therefore, the SI protected skeletal muscle cells from oxidative damage, attributing to its antioxidant activity.Study 2 was to investigate the protective effect of genistein(GEN) on antioxidative defense system and lipid peroxidation in chick SMC. Chick SMC were treated with 0, 1,4, and 16μmol/L GEN, respectively. The activities of GSHPx, T-SOD and CAT and the concentration of malondialdehyde (MDA) were measured in supernatant of the medium for culture of chick SMC. Meanwhile the expression of GSHPx mRNA was determined by RT-PCR method. Compared to control, treatment with 1,4 or 16μmol/L GEN decreased MDA production yield by 33.85%(p<0.05),33.08%(p<0.05) and 50.77%(p<0.05), respectively. The activity of SOD significantly increased after treated with 1μmol/L or 16μmol/L GEN (p<0.05). The addition of GEN at 4 or 16μmol/L improved the activity of GSHPx (p<0.05). These results indicated that GEN could inhibit lipid peroxidation and improve antioxidative property by inhibiting MDA production and increasing the activities of antioxidant enzymes in chick SMC.Study 3 was conducted to investigate the protective effect of GEN and SI (from 20 to 80μmol/L) on antioxidative defense system and membrane fluidity in chick SMC treated with 50μmol/L H2O2/FeSO4 for 48h. GEN and SI supplementation significantly increased the activities of T-SOD and GSHPx (p<0.05) and decreased the MDA production yield (p<0.05), regardless of H2O2/FeSO4 treatment. GEN at 80μmol/L and SI at 40,80μmol/L decreased the secretion of CK in H2O2/FeSO4 treated cells (p<0.05 or p<0.01). Fluorescence polarization values and micro viscosities observed in H2O2/FeSO4 treated cells were significantly higher than those observed in untreated cells. The addition of 80μmol/L GEN or SI decreased the fluorescence polarization value in H2O2/FeSO4 treated cells (p<0.05). The microviscosity value decreased significantly due to GEN or SI addition (p<0.05). Adding 20μmol/L GEN or SI improved the expression of GSHPx mRNA. These results suggested that GEN or SI has antioxidative effects in SMC.Study 4 was aimed at investigating the effects of SI on growth performance, meat quality, and antioxidation in male broilers. A total of 1500 42-day-old birds were allotted to five treatments with six replicates per treatment (50 birds per replicate). Birds were fed diets supplemented with 0,10,20,40, or 80 mg/kg SI, respectively, for a period of 3 weeks ad libitum. The results showed that dietary supplementations with 10 mg/kg or 20 mg/kg SI increased weight gain by 13.6% and 16.2%(p<0.01), and elevated feed intake by 7.37%(p<0.05) and 11.2%(p<0.01), respectively. Addition of 10mg/kg SI decreased feed:gain by 5.5%(p<0.05). Supplementation with SI in the diet increased water holding capacity (WHC) 24h,72, and 96 postmortem (p<0.01). The addition of 40 mg/kg SI significantly increased the pH value of meat 24h and 96 postmortem (p＜0.01). Adding 40mg/kg SI decreased meat color L value 45min and 96 postmortem (p<0.05). Lactic acid (LD) production yield was significantly decreased in breast muscles of 10,20, or 40mg/kg SI-supplemented chickens 45min postmortem (p<0.01). MDA concentration was reduced in plasma of 10 or 20 mg/kg Si-supplemented chickens (p<0.05), in liver of SI-supplemented chickens (p<0.01), and in breast meat of 20,40, or 80 mg/kg SI-supplemented chickens 45min and 72h postmortem (p<0.05 or p<0.01). The addition of 40 or 80mg/kg SI significantly increased TAOC (p＜0.01) and slightly elevated T-SOD activity (p<0.1) in plasma of chickens. The dose of 80mg/kg SI slightly improved catalase activity in plasma (p<0.1). In breast muscle, dietary SI caused a decrease in T-SOD activity 45min postmortem (p<0.05 or p＜0.01), while increased T-SOD activity 12h,48h and 72h postmortem (p<0.05 or p＜0.01). CAT activity significantly increased 70.61% by the supplemental SI at 40 mg/kg level (p<0.05). The results indicated that dietary SI could improve growth performance and meat quality by decreasing lipid peroxidation and enhancing antioxidative capacity in male broilers.The study 5 was designed as a 2 x 2 factorial arrangement with 2 types of fish oil (oxidized oil and fresh oil) and 2 SI supplementation levels (0 and 20 mg/kg) to determine the effect of dietary SI on performance, meat quality and antioxidative property in male broilers. Six replicates of 45 birds (42 d old) were used for each treatment. The experiment lasted for 3 weeks. The results showed that average daily gain, feed intake, feed conversion and carcass traits were not affected by SI supplementation or fish oil types. The oxidized fish oil treatment significantly increased breast muscle color L value (p<0.01) and decreased a*(p<0.01) and b* values (p＜0.01). The activity of CK and TAOC in plasma decreased by oxidized fish oil treatment (p<0.01). Oxidized fish oil treatment increased MDA concentration and reduced TAOC in breast muscle (p<0.01). The SI supplementation decreased MDA and LD concentrations (p<0.05 or p＜0.01), and improved the activities of T-SOD, GSHPx and TAOC (p<0.05 or p<0.01). The SI supplementation increased the expression of GSHPx mRNA. The interaction effect of fish oil and SI supplementation was significant on the concentrations of MDA and LD and the activities of CAT, GSHPx and TAOC in breast muscle (p<0.05 or p<0.01). The results indicated that oxidized fish oil treatment induced lipid peroxidation and reduced breast muscle color oxidant stability. Dietary SI could improve antioxidative property of meat in male broilers fed oxidized fish oil diet.It was concluded from studies 1,2 and 3 that dietary SI and GEN are effective in protecting SMC from oxidative damage. Studies 4 and 5 suggested that there may be potential for soy isoflavones to inhibit lipid oxidation, improve meat quality by ameliorating the antioxidant status and regulating antioxidant enzyme mRNA expression in vivo.更多还原