【作者】 张玉俊；

【导师】 林海；

【作者基本信息】 山东农业大学 ， 动物营养与饲料科学， 2011， 硕士

【摘要】 为对糖皮质激素调控肉仔鸡能量食欲的mTOR通路进行探索,本研究利用人工导入外源糖皮质激素的方法模拟应激,从应激发展的两个阶段——急性和长期过程着手,分别分析了下丘脑mTOR通路相关基因和食欲相关基因表达量的改变,并分析了下丘脑中mTOR通路相关蛋白的磷酸化程度,为应激状态下下丘脑能量食欲调控模式的建立和改善实际生产中应激引起的能量浪费提供理论依据。第一部分,我们主要对长期注射DEX所引发的应激进行研究,共分两个试验。试验一0日龄AA (Arbor Acres)雄性肉仔鸡120只,正常饲喂至4日龄。4日龄起,试验动物在每天的2:00-3:00, 8:00-9:00,14:00-15:00,20:00-21:00进行饲喂,直至试验结束。Trial 1选取体重相近的11日龄AA雄性肉仔鸡40只,平均分为A、B两个组。每组设10个重复,每个重复2只鸡。11日龄起早6:00,A组按2.6 mg/kg BW的剂量皮下注射地塞米松磷酸钠,B组同剂量皮下注射生理盐水,连续注射三天。13日龄8:00和9:00两个时间点分别从每个重复取1只鸡,静脉采血后,颈部移位屠宰,迅速剥离下丘脑液氮保存,待RNA提取。血液样品3000rpm离心10min后取血浆,冷冻保存待测。Trial 2分组同trial 1,11日龄起注射时间为每天10:00(空腹1h), 13日龄的12:00(处理2h,空腹3h)采样,样品采集同trial 1。试验主要结果显示:糖皮质激素在采食前即8:00(处理2h,空腹5h)对mTOR通路各个因子的mRNA相对表达量均未出现显著影响;恢复饲喂1h后(9:00),糖皮质激素显著上调了PI3Cα的mRNA相对表达量,且糖皮质激素组PI3Cβ,EEF2也有上调的趋势。对其它因子的影响差异并不显著。Trial 2的结果表明糖皮质激素在12:00(处理2h,空腹3h)对肉仔鸡下丘脑中各个因子mRNA的相对表达量的影响均不显著。试验二将AA雄性肉仔鸡正常饲喂至4日龄,4日龄后按试验一的方式用正常日粮对试验动物进行分段饲喂。将体重相近的9日龄AA雄性肉仔鸡48只,平均分为A、B、C、D四个组。9日龄起A、B两组分段饲喂高能日粮(代谢能,3.6kcal/kg;粗蛋白,20%),C、D两组分段饲喂低能日粮(代谢能,2.6kcal/kg;粗蛋白,20%),试验在单饲笼内进行,每组设6个重复,每个重复2只鸡。11日龄起每天6:00,A、C组按2.6 mg/kg BW的剂量皮下注射地塞米松磷酸钠,B、D两组皮下注射同剂量的生理盐水,连续注射三天。13日龄早8:00(取样后A、B饲喂高能日粮,C、D两组饲喂等量低能日粮)和9:00两个时间点分别从每个重复取1只鸡,静脉采血后,颈部移位屠宰,迅速剥离下丘脑,待RNA提取。血液样品3000rpm离心10min后取血浆,冷冻保存待测。试验结果表明:低能日粮采食条件下糖皮质激素对肉仔鸡采食前、后下丘脑中NPY, GR, INSR, AKT3, 4EBP1, mTOR, AgRP, POMC的mRNA相对表达量影响均不明显。糖皮质激素处理使低能日粮组试验动物采食后GR mRNA相对表达量有下调趋势,AgRP mRNA相对表达量有上调的趋势。高能日粮采食条件下,糖皮质激素注射组显著的上调了采食前肉仔鸡下丘脑中INSR, AKT3, 4EBP1的mRNA相对表达量(P<0.05),而POMC mRNA相对表达量有下调的趋势,上述变化在采食后均消失。糖皮质激素对高能日粮采食前、后丘脑中NPY, GR, mTOR, AgRP mRNA相对表达量的影响差异均不显著(P>0.05)。第二部分,将体重相近的10日龄AA雄性肉仔鸡96只随机分为六组,每组8个重复。10日龄早9:00对所有试验肉仔鸡禁食处理,11日龄早6:00按A、B、C(处理组),A’、B’、C’(对照组)进行处理,11日龄9:00,将A,A’两组处死取样,同时B、B’两组饲喂高能日粮,C、C’两组饲喂低能日粮,10:00对剩余试验肉仔鸡取样。试验结果显示:糖皮质激素单次注射对空腹状态下肉仔鸡下丘脑中AgRP,NPY, POMC, GR,Ghrelin,MC4R,INSR的mRNA相对表达量均没有显著影响(P>0.05)。恢复饲喂1h高能日粮后,糖皮质激素显著上调了下丘脑中NPY mRNA的相对表达量(P<0.01),但糖皮质激素在上述条件下对肉仔鸡下丘脑中AgRP, POMC, GR, Ghrelin, MC4R, INSR的mRNA相对表达量影响均不显著(P>0.05);恢复饲喂低能日粮后,糖皮质激素使肉仔鸡下丘脑中NPY mRNA的相对表达量有上调趋势,但该条件下AgRP, POMC, GR, Ghrelin, MC4R, INSR的影响均不显著(P>0.05)。更多还原

【Abstract】 To investigate the effects and mechanisms of glucocorticoids on the mammalian target of rapamycin (mTOR) appetite and energy pathway of broilers (Gallus gallus domesticus), exogenous glucocorticoids was used to mimick stress on two phases of stress development of broilers. Changes of Protein and mRNA expression level of important genes in appetite regulation and mTOR pathway in hypothalamus were measured and analyzed, in order to providing countermeasures for reforming energy waste in broiler chicken production. In experiment 1, there were two similar trails. In trial 1, 80 male AA broilers were divided into two groups: glucocorticoids and control. Each group had 10 repetitions and each repetition had 4 broilers. Broilers were injected with the same dose of dexamethasone(DEX) and saline at 6:00 (11d-13d), respectively. At the day of 13, samples (blood and hypothalamus) were obtained at 8:00 and 9:00. In trial 2, group dividing and the treatment were the same as which in trail 1, except that the time of injection was 10:00, and samples were obtained at 12:00. The results showed that the glucocorticoids had no significant effect on the relative expression of mTOR pathway-related genes at 8:00(before foodintake). After refeeding 1h, glucocorticoids significantly increased the relative expression of PI3Cα, and tend to increase the relative expression of PI3Cβand EEF2. But no sinificant effect on the other factors. Compared with control treatment, the mRNA level of mTOR pathway-related genes were no significant changes in trial 2.In experiment 2, 48 male AA broilers with similar body weight were divided into 4 groups randomly, there were 6 repetitions in every group with 2 broilers in every repetition, rearing in separate cage. In group 1, male AA broilers were subcutaneous injected with DEX (2.6 mg/kg BW) at 6:00 from 11 to 13 days old ,and diets were high energy (ME, 3.6kcal/kg; CP, 20%), Group 2 is control group, Saline was injected with a suitable dose ,and diets were high energy, treats were the same as in group 1 except that dexamethasone was displaced by saline. Group 3 animals were injected by DEX and diets were low energy(ME, 2.6kcal/kg; CP, 20%), in group 4, animals were injected by saline, and diets were low energy. At the day of 13, samples (blood and hypothalamus) were obtained at 8:00 and 9:00 (GroupA and B were feed by high energy food, and Group C and D were feed by the same quality low energy food with Group A and B). The results showed that glucocorticoids had no significant effect on the relative expression of NPY, GR, INSR, AKT3, 4EBP1, mTOR, AgRP, POMC before and after ingestion in the stage of low energy diets. The glucocorticoids tend to reduce relative expression of GR, and tend to increase the relative expression of AgRP, in the low energy diet groups. In the stage of high energy diets, the glucocorticoids increased the relative expression of INSR, AKT3, 4EBP1(P<0.05), and the relative expression of POMC trend to decrease. All the effects vanished after refeed. there was no significant effect of glucocorticoids on relative expression of NPY, GR, mTOR, AgRP before and after ingestion in the stage of high energy diets.In part 2, The broilers were randomly assigned to 6 group, 8 birds per group. After 21h fasting at 6:00, three groups (A, B, C) were injected by DEX in a dose of 2.6 mg/kg.BW, and another three group(A’, B’, C’) were treated with same dose saline. At the day of 11, blood and hypothalamus of Group A and A’were obtained at 9:00, at the same time the animals of Group B and B’were feed by high energy diets, the animals of Group C and C’were feed by low energy diets. At 10:00 all the blood samples were obtained, and hypothalamus were quick-freezed in liquid nitrogen. The results showed that glucocorticoids had no significant effect on the relative expression of AgRP, NPY, POMC, GR, Ghrelin, MC4R, INSR after fasting for 24h. The glucocorticoids significant increased the relative expression of NPY(P<0.01), and no significant changes of AgRP, POMC, GR, Ghrelin, MC4R, INSR were found. In the DEX-treated group the relative expression of NPY trend to increase at the stage of 1h refeeding of low energy diets, and there were no significant changes of the relative expression of AgRP, POMC, GR, Ghrelin, MC4R, INSR.更多还原