【作者】 袁丽霞；

【导师】 赵茹茜；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2009， 博士

【摘要】 本文首先通过比较蛋鸡和肉鸡下丘脑食欲(appetite)和能量稳态(energy homeostasis)调节相关基因的表达,发现促肾上腺皮质激素释放激素(CRH)、糖皮质激素受体(GR)、脂肪和肥胖相关(FTO)基因的表达在品种间存在差异。然后检测了蛋鸡和肉鸡种蛋内的激素水平,发现肉鸡蛋内含有较高的瘦素(1eptin)和皮质酮。根据以往的研究报道,提出蛋中leptin可能通过影响下丘脑食欲和能量稳态调节相关基因的表达对后代生长性能产生程序化影响。为验证这一假说,设计了蛋内注射leptin及其拮抗剂的试验,观察对肉鸡早期生长发育影响的同时,下丘脑食欲和能量稳态调节相关基因表达的变化,为揭示母源性leptin对禽类生长程序化影响的机制提供实验证据。1蛋鸡和肉鸡下丘脑食欲和能量稳态调节相关基因表达的比较研究蛋鸡和肉鸡在体重和身体组成上存在明显的差异,而这种差异形成的机制还不清楚。本试验通过对出雏后7日龄(D7)蛋鸡和肉鸡的比较发现,肉鸡具有较高的体重和采食量(P<0.001),但单位体增重的采食量(饲料转换比,FCR)反而蛋鸡较高(P<0.001)。采用原位杂交和(或)real-time RT-PCR的方法发现下丘脑中的一些基因如神经肽Y(NPY)、刺鼠相关蛋白(AGRP)、阿黑皮素原(POMC)、食欲素(ORX)、leptin受体(LEPR)和20-羟基类固醇脱氢酶(20HSD)的mRNA表达在品种间都没有显著差异(P>0.05)。下丘脑的乙酰辅酶A羧化酶(ACC)和脂肪酸合酶(FAS)的mRNA表达量肉鸡有高于蛋鸡的趋势(P<0.10)。肉鸡下丘脑中CRH mRNA (P<0.05)的表达较蛋鸡低,但蛋白水平正好相反(P<0.01),表明CRH在转录、翻译和分泌等不同水平的调节过程中存在差异。肉鸡下丘脑中GR mRNA (P<0.05)和蛋白(P<0.01)水平均显著高于蛋鸡,说明肉鸡下丘脑中由GR介导的负反馈调节CRH转录和释放功能的增强。此外,FTO基因在肉鸡的下丘脑中高表达(P<0.01)。以上结果揭示了与能量平衡和肥胖相关的基因如GR、CRH、FTO的表达受到遗传选育的影响,并且可能对鸡的体重和长期能量稳态起到重要的调节作用。2蛋鸡和肉鸡下丘脑CRH、GR基因启动子的甲基化分析及蛋内激素水平的比较本试验通过比较蛋鸡和肉鸡下丘脑CRH、GR基因启动子的甲基化水平以及两品种鸡种蛋内的leptin、皮质酮的水平,来观察CRH和GR基因表达的品种差异是否伴随DNA甲基化的差异,并观察蛋内激素水平在品种间是否不同。甲基化敏感限制性内切酶PCR法的研究发现,CRH和GR基因启动子片段的甲基化水平在品种间没有差异(P>0.05)。放射免疫分析法(RIA)和酶免疫分析法(EIA)的结果显示肉鸡蛋黄中leptin以及皮质酮的水平均显著高于蛋鸡(P<0.05)。以上结果提示：蛋中的母源性激素如leptin、皮质酮在品种间存在差异,但这种差异是否与生长和基因表达的品种差异有关还有待进一步研究。3蛋内注射leptin对雏鸡下丘脑食欲和能量稳态调节相关基因表达的影响为了探讨母源性leptin对三黄肉鸡育雏期下丘脑食欲和能量稳态相关基因表达的影响,本试验挑选蛋重相近的三黄肉鸡种蛋,随机分为三组：蛋清内注射100μL溶剂(PBS)的对照组(control, Con), leptin处理组分别注射0.5μg/100μLPBS(低剂量组,LL)和5.0μ/100 pL PBS(高剂量组,HL)的重组小鼠leptin,出雏后饲养至21日龄时采取样品。蛋内注射leptin影响肉鸡出雏后的早期生长,并呈现性别特异性和剂量依赖性。注射高、低剂量的leptin均使得肉鸡出雏重下降,特别是低剂量组母鸡(P<0.01)。育雏期处理组肉鸡的生长速度高于对照组,21日龄时处理组肉鸡的体重显著高于对照组(P=0.042,P=0.003). Real-time RT-PCR和Western blotting方法检测发现,leptin注射影响21日龄肉鸡下丘脑基因和蛋白的表达,高剂量组公鸡表现更为明显。除了CRH mRNA表达没有变化外,高剂量组公鸡下丘脑中LEPR、FAS、GR的mRNA表达均显著下调(P<0.05)。Leptin处理组公鸡下丘脑中FTO mRNA的表达均极显著地升高(P<0.01)。母鸡只有低剂量组GR mRNA的表达显著下降(P<0.05)。公鸡下丘脑中CRH蛋白含量在高剂量组显著升高(P<0.05),GR蛋白水平在高、低剂量组均极显著升高(P<0.01),而母鸡无显著变化(P>0.05)。原位杂交的结果显示,GR mRNA广泛分布于21日龄肉鸡脑的各个区域,主要是大脑纹状体、丘脑的圆核(ROT)、背外侧核(DLN)、卵圆核(OV)、上网状核(Rsd)等,在下丘脑视前区(POA)也有较多分布,其次是下丘脑外侧区(LHy)、室旁核(PVN)、室周核(PHN)、腹内侧核(VMN)、漏斗核(IN)和海马(Hp)等。其中高、低剂量leptin均使得PVN.PHN和VMN中GR mRNA的表达下调(P<0.05),而对LHy、IN和Hp的GR mRNA影响不显著(P>0.05)。以上结果提示：蛋内注射1eptin影响肉鸡育雏期的生长发育,改变下丘脑食欲和能量稳态调节相关基因的表达,特别是GR的水平。4蛋内注射leptin及其拮抗剂对雏鸡下丘脑食欲和能量稳态调节相关基因表达的影响为了进一步探讨母源性leptin对三黄肉鸡育雏期下丘脑食欲和能量稳态调节相关基因表达的影响机制,本试验挑选蛋重相近的三黄肉鸡种蛋,随机分为四组：对照组(100μLPBS, Con)、leptin处理组(0.5μg,L)、leptin拮抗剂组(0.5μg,LA)和leptin+leptin拮抗剂组(各0.5μ,L+LA)。出雏后饲养至21日龄时进行采样。蛋内注射leptin及其拮抗剂影响肉鸡出雏后的早期生长和下丘脑中相关因子的水平,并呈现性别差异。蛋内注射leptin及其拮抗剂后,肉鸡的出雏重和Con组相比无明显差异(P>0.05)。无论公母,和其它三组相比,蛋内注射leptin显著提高了肉鸡育雏期的体重、相对体增重(P<0.05),也提高了育雏期肉鸡的采食量,和Con组相比差异显著(P<0.05)；两个拮抗剂组和Con组相比,体重、采食量等差异不明显(P>0.05)。RIA的结果显示,21日龄时LA、L+LA组母鸡血清中leptin的水平显著高于Con组和L组(P<0.05)。Real-time RT-PCR和Western blotting方法检测发现,21日龄时L组母鸡的GR mRNA表达显著低于Con组(P<0.05)。L+LA组公鸡LEPR mRNA显著高于Con组(P<0.05),而CRH mRNA水平反而低(P<0.05)。蛋白水平只有L组公鸡的GR升高,显著高于Con组(P<0.01)和L+LA组(P<0.05)。荧光原位杂交(FISH)的结果显示,和Con组相比,母鸡21日龄时L组PVN、PHN中GR mRNA的表达下调(P<0.05),L+LA组的GR mRNA在PHN(P<0.01)和LHy(P<0.05)表达下调。以上结果基本上重复了leptin蛋注的试验,并证明了蛋内注射leptin可程序化的影响肉鸡出雏后的早期生长发育。HPA轴的CRH和GR参与leptin的这种程序化作用,从而对鸡的体重和能量稳态进行调节。更多还原

【Abstract】 The present study first demonstrated the difference in hypothalamic expression of genes regulating appetite and energy homeostasis, such as corticotropin-releasing hormone (CRH), glucocorticoid receptor (GR) and fat mass and obesity associated (FTO) gene, between layer and broiler chicks. Secondly, the leptin and corticosterone contents in the egg were also different between breeds, which were higher in the yolk of broilers. To test the hypothesis that maternal leptin play an important role in posthatch growth and energy homeostasis, leptin and its antagonist were administered in ovo to investigate the programming effect on early posthatch growth, as well as hypothalamic expression of genes regulating appetite and energy homeostasis, especially CRH and GR.1 Comparative studies of the appetite and energy homeostasis regulation genes in the hypothalamus of layer and broiler chicksLayer and broiler chickens demonstrate striking differences in body weight and body composition. However, the mechanism underlying such difference is still elusive. The body weight and food intake were already significantly higher in broiler chicks at day (D) 7 posthatching, but the food intake relative to body weight gain (food conversion ration, FCR) was actually higher in layer chicks. No breed differences were observed for hypothalamic expression of neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin (POMC), orexin (ORX), leptin receptor (LEPR) and 20-hydroxysteroid dehydrogenase (20HSD) at D7 by in situ hybridization (ISH) and/or real-time RT-PCR. A tendency higher expression of hypothalamic acetyl CoA carboxylase (ACC) or fatty acid synthase (FAS) mRNA was found in broilers than layers (P<0.10). Broiler chicks expressed lower CRH mRNA (P<0.05) yet higher accumulation of CRH peptide in hypothalamus, which means the different levels in transcription, translation and release of CRH. However, broiler chicks expressed significantly higher GR mRNA (P<0.05) and protein (P<0.01) in hypothalamus compared to layer chicks, suggesting an augmented GR-mediated negative feedback regulation of CRH transcription and release in broiler chicks. Furthermore, FTO gene was also more highly expressed in hypothalamus of broiler chicks (P<0.05). These results suggest that the genes related to energy balance and obesity, such as GR, CRH and FTO, rather than orexigenic neuropeptides, are impacted by the genetic selection practices and play a role in the regulation of body weight and energy homostasis in chicken.2 Analysis of the methylation levels of CRH and GR gene promoters in hypothalamus and egg hormone contents in layer and broler chicksTo investigate whether the gene expression differences were related to the DNA methylation and associated with breed dfferences in maternal hormone deposition in the egg, the methylation levels of CRH and GR gene promoter in the hypothalamus, and the leptin and corticosterone contents in the egg were determined in layer and broiler chickens. No difference was observed in the hypothalamic CRH or GR gene promoter methylation levels (P>0.05) between layer and broiler chicks by methylation-sensitive restriction enzyme PCR. Radioimmunoassay (RIA) and enzyme immunoassay (EIA) methods showed higher levels of yolk leptin and corticosterone in broiler eggs than layer eggs (P<0.05). The results suggested that the maternal hormones, such as leptin and corticosteron, are different in breeds. However the further research should be continued to identify the relationship between maternal hormones and breed differences.3 Effects of in ovo leptin administration on the appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicksLeptin in ovo administration was employed to investigate the effect of leptin deposited in eggs on the expression of appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicks. Breeder eggs were randomly allocated to three groups and injected with 0.5μg (low leptin, LL),5.0μg (high leptin, HL) of recombinant mice leptin in 100μL of PBS or 100μL PBS (Control, Con) before incubation. The hatchings were raised under the same condition till 21 days (D21) of age when the samples were collected for analysis. The early posthatch growth rate of broiler chicks was influenced by leptin which showed gender specific and dose dependent. Chicks treated with high or low doses of leptin in ovo had lower hatch weight than the control group, especially the female chicks in low leptin group (P<0.01). The posthatch growth rate was higher in both leptin-treated groups compared to control group. At D21, chicks treated with leptin in ovo demonstrated significantly higher body weight than control group (P=0.042, P=0.003). The hypothalamic gene and protein levels of 21-old-day chicks were measured by real-time RT-PCR or Western blotting. Except for the CRH mRNA, the expressions of hypothalamic LEPR, FAS and GR mRNA were decreased in male chicks of HL group (P<0.05). Significantly higher FTO mRNA (P<0.01) levels were detected in the hypothalamus of male chicks in both leptin groups. In the hypothalamus of female chicks, GR mRNA levels were decreased in LL group (P<0.05). The CRH protein levels were higher in the hypothalamus of male chicks in HL group (P<0.05). Hypothalamic GR protein levels were significantly elevated by both low and high doses of leptin in ovo (P<0.01). No changes were found in female chicks. The GR mRNA was widely distributed in the brain of D21 broilers by in situ hybridization, mainly in the corpus striatum, nucleus rotundus (ROT), dorsolateral nucleus (DLN), nucleus ovoidalis (OV), nucleus reticularis superior (Rsd) and the preoptic area (POA) of hypothalamus. The GR mRNA signals was also detected in lateral hypothalamus (LHy), paraventricular nucleus (PVN), periventricular nucleus (PHN), ventromedial nucleus (VMN), infundibular nucleus (IN) and hippocampus (Hp). In high or low doses of leptin group, the expressions of GR mRNA were found down regulated in PVN, PHN and VMN (P<0.05) of D21 broiler chicks. However, no difference was found in LHy, IN or Hp of D21 broiler chicks among groups (P>0.05). These results indicate that in ovo administration of leptin influence the early posthatch development of broiler chicks, and the expressions of appetite and energy homeostasis regulation genes, especially the GR.4 Effects of in ovo leptin and its antagonist administration on the appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicksLeptin and its antagonist in ovo administration were employed to further investigate the effect and mechanism of leptin deposited in eggs on the appetite and energy homeostasis regulation genes in the hypothalamus of broiler chicks. Breeder eggs were randomly allocated to four groups and injected with recombinant mice leptin (0.5μg, L), recombinant mice leptin antagonist (0.5μg, LA), leptin (0.5μg) plus leptin antagonist (0.5μg) (L+LA) in 100μL of PBS or 100μL PBS (Control, Con) before incubation. The hatchings were raised under the same condition till 21 days (D21) of age when the samples were collected for analysis. The early posthatch growth rate of broiler chicks was influenced by leptin and its antagonist which showed gender specific. All treated group chicks had no difference on hatch weight with the control group (P>0.05). Compared with other groups, the posthatch body weight and relative body weight gain were elevated in leptin group of both male and female broiler chicks (P<0.05). The food intake of broiler chicks was increased in leptin group, and significantly higher than control group (P<0.05). However, the antagonist had no effect on the body weight and food intake compared with control group chicks. Using RIA, higher serum leptin levels were detected in female chicks of both LA and L+LA group than contol and leptin group at D21 (P<0.05). The expressions of LEPR, CRH and GR mRNA or protein in the hypothalamus of broiler chicks were measured by real-time RT-PCR and Western blotting. At D21, female chicks in leptin group showed lower expressions of GR mRNA than control group (P<0.05). The LEPR mRNA levels were higher in male chicks of L+LA group than control group, wheras the CRH mRNA levels were lower. Only the GR protein levels were found elevated in male chicks of leptin group than control (P<0.01) and L+LA group (P<0.05). Furthermore, at D21 female chicks, the GR mRNA levels were decreased in PVN and PHN of leptin group compared with control group (P<0.05), wheras the GR mRNA levels were down regulated in PHN (P<0.01) and LHy (P<0.05) of L+LA group by fluorescence in situ hybridization (FISH). These results indicate that leptin has the effect on the early posthatch development of broiler chickens. CRH and GR play an important role in the programming effect of leptin, and regulate the body weight and energy homeostasis of chickens.更多还原