【作者】 胥清富；

【导师】 陈杰；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2002， 博士

【摘要】 一、二花脸猪和大白猪生长及生长轴有关激素和受体基因表达的发育性变化 为了探讨动物生长发育的神经内分泌及分子生物学机理，本研究以生长慢的纯种二花脸猪和生长快的纯种欠白猪为动物模型，用相对定量RT-PCR方法和RIA法，对出生后（0、3、20、30、45、90、120、180日龄）二品种猪肝脏和背最长肌的GHR、IGF-I及IGF-IR mRNA水平、组织中IGF-I含量以及血清中瘦蛋白。T₃、T₄，胰岛素和胰高血糖素浓度进行了系统的比较研究，结果如下： 1、体重增长及肝脏和肌肉器官生长规律：从出生到180日龄大白猪体重和平均日增重高于二花脸猪；大白猪肝脏重量和平均日增重高于二花脸猪，但二品种肝脏相对重量无明显差异；大白猪背最长肌重量、相对重量和平均日增重均高于二花脸猪；二花脸猪体重、平均日增重、肝脏和肌肉绝对重量、相对重量和平均日增重均无明显的性别差异。 2、血中几种激素水平的发育性变化：二花脸猪和大白猪（1）血中瘦蛋白水平发育性变化模式一致，30日龄前较低，90～180日龄维持在较高水平，总体上二花脸猪高于大白猪，雌性高于雄性；（2）血清中T₃和T₄水平发育性变化模式基本相同，T₃无明显的品种和性别差异，而二花脸猪T₄高于大白猪，但无性别差异；（3）血清中胰岛素发育性变化模式不同，二花脸猪0～120日龄随日龄增加呈上升趋势，随后呈下降趋势，而大白猪0～20日龄增加，随后无明显变化。总体上二花脸猪明显高于大白猪，雄性高于雌性；二品种猪血清中胰高血糖素水平的发育性变化模式相同，但无明显品种和性别差异。 3、肝脏GHR、IGF-I和IGF-IR基因表达发育性变化：（1）二品种猪肝脏GHR mRNA水平发育性变化模式基本一致，即从出生到120日龄肝脏GHR丰度随年龄增加而增力，120日龄后维持在较高水平；30日龄前同日龄比较差异不明显，90、120、180日龄时大白猪高于二花脸猪；总体上大白猪高于二花脸猪（P＜0.01），但雌性和雄性二花脸猪差异不明显（P＞0.05）；（2）二品种猪肝脏IGF-I mRNA水平发育性变化与GHR mRNA相似，随日龄增加而增加，二者呈明显正相关（R=0.70，P＜0.01），其中大白猪R=0.58（P＜0.01），二花脸猪R=0.75（P＜0.01）；肝组织中IGF-I含量发育性变化规律与GHR和IGF-I mRNA一致，与二者均呈明显正相关，其中与GHR mRNA相关系数为R=0.67（P＜0.01），与IGF-I mRNA相关系数R=0.78（P＜0.01）；肝脏GHR（R=0.95，P＜0.01），IGF-I mRNA（R=0.91，P＜0.01）丰度及肝组织IGF-I含量（R=0.97，P＜0.01）均与动物平均日增重呈明显正相关；总体上大白猪肝脏IGF-I mRNA水平（P＜0.01）及组织IGF-I含量（P＜0.05）均高于二花脸猪；提示肝脏IGF-I的合成由GHR介导，二者在猪生长发育中起重要作用。（3）肝脏IGF-IR mRNA无明显的年龄、 臀清富 生长激素作用的靶基因在猪肝脏和肌肉上的表达及调控品种和性别差异（P＞0．05）。 4、背最长肌 GHR、IGFI和 IGFIR基因表达发育性变化：（l）二花脸猪和大白猪背最长肌 GHR mRNA相对丰度发育性变化模式基本一致，随年龄增加有增加的趋势，其中二花脸猪在20和90日龄有两峰值，30，120～180日龄有所下降，而大白猪在20～90日龄随日龄增力。而增加，随后维持在较高水平；总体上大白猪高于二花脸猪（P＜0．05）；（2）H品种间背最长肌＊F二 相对丰度的发育性变化模式相似，0～30日龄较低且无明显变化，30～90日龄显著增加，随后保持在较高水平Z 二品种背最长肌组织中＊F－I含量从出生（0天）到90日龄呈上升趋势；随后呈下降趋势；总体上二品种1＊PlmRNA无明显差异（P＞0刀5），但组织中1＊卜1含量大白猪高于二花脸猪（P＜0．05）；提示月几肉＊F－I基因表达有转录后调节；GHR二RNA相对丰度与＊F－I＜ RNRNA（＊一0．忙，P刃刀1）和组织中IG卜1含量（＊一O．69，＊＜0．01）明显正相关，提示背最长肌 GHR也参与介导组织局部 IGF－I的合成；（3）IGF－IR mRNA无明显发育性变化（P＞0刀5），但总体上大白猪高于二花脸猪（P＜O刀5）；（4）二花脸猪背最长肌GHR、IGF－I和＊F－IR ＞RNA均无明显的性别差异（P＞0．05）。二、重组生长激素对猪生长、血清有关激素水平肌脏和肌肉GHR和！GFJ基因表 达的影响 16头去势长白 X大白杂交公猪，随机分成试验组和对照组，每组 8头。试验组肌肉注射pGH卜 mg．d“’)，而对照组注射等体积生理盐水。28天后屠宰采样，用相对定量 RT－PCR方法坝11定肝脏、背最长j几和半侧几 GHR和＊F－IIRNA丰度；并用 RIA法测血中有关激素浓度。结果显示：（1）与对照组相比，试验组平均日增重提高21．6％（P＜0．05），血清＊F－I水平提高70．94O（P＜0．of），胰岛素提高58．90（P＜0．05）；IL－2水平的有增力。的趋势（提高16）％，P＝O．14)，而T3、T4、胰高血糖素无明显变化（P＞0．05）。门)试验组肝脏 GHR、IGF。ImRNA相对丰度和肝组织中＊F－I含量分别提高24．凸％（P＜O．05），巧30％（P＜0刀1）和2引％（P＜0．01）；（3）试验组背最长肌GHR（P＞0．05）和＊F－I＞RNA（P＞0．05）相对丰度无明显变化，而背最更多还原

【Abstract】 1 Studies on Growth and Developmental Changes of Serum Level of Some Hormones and Expression of Target Genes for Growth Hormone Action in Erhualian pigs and Large White pigsIn order to research the neuroendocrinological and molecular biology mechanism of animal growth and development, purebred male Large White (LW), male and female Erhualian (EHL) pigs with significant differences in growth rate were employed in present study to investigate the developmental changes of serum levels of some hormones and expression of target gene for growth hormone action in the liver and skeletal muscle. The animals were fed according to the breeding standards of USA NRC and Chinese Local Pigs respectively. Four piglets of each breeds or sexes (EHL) were slaughtered at 0, 3, 20, 30, 45, 90, 120 and 180 day respectively for liver, longissimus dorsi (LD) muscle and blood sampling. Serum levels of leptin, insulin, glucagon, T3 and T4 were determined by RIA. The total RNA of liver and LD muscle was extracted and the abundance of liver growth hormone receptor (GHR), insulin-like growth factor 1(IGF-I) and IGF-IR messenger ribonucleic acid (mRNA) was determined by relative quantitative reverse transcription polymerase chain reaction (RT-PCR) with 18S rRNA as internal standard. Results showed as follows:1.1 Developmental changes of body, liver and LD weight gain in EHL and LW pigs. From day 0 to day 180, the body weight (BW), average daily body weight gain (ABWG), liver weight, average daily liver weight gain (DLWG), LD muscle weight, average daily LD weight gain (DLDWG) and rate of LD weight to body weight of male LW at each age were higher than those of male EHL. However, no significant differences of relative liver weight to body weight between male EHL and LW pigs, BW, ABWG, liver weight, LD weight, rate of liver and LD weight to BW, DLWG and DLDWG between male and female EHL pigs were found.1.2 Developmental changes of serum levels of some hormones in EHL and LW pigs. (1) The pattern of developmental changes of serum level of leptin in EHL and LW pigs were similar, which being low before day 30 and high from day 90 to day 180 in both breeds. The serum leptin concentration was higher in male EHL pigs than in male LW pigs (P<0.01), and in female than in male EHL (P<0.01). (2) Similar developmental changes of serum level of T3 or T4 in EHL and LW pigs were also observed. There was higher serum level of T4 (P<0.05) but T3 (P>0.05) in male EHL pigs than in male LW pigs, while both T3 (P>0.05) and T4 (P>0.05) were not markedly different between male and female EHL pigs. (3) The serum insulin concentration in male EHL pigs had a potential increase from day 0 to day120 and then potential decrease, while in male LW it had a potential increase from birth to day 20 and no significant changes thereafter. The serum level of insulin in male LW was lower than that in EHL (P<0.01), while there was no detectable difference of serum glucogan level between breeds (P>0.05)1.3 Developmental changes of hepatic GHR, IGF-I and IGF-IR mRNA abundance in EHL and LW pigs. (1) The pattern of developmental changes of relative abundance of hepatic GHR mRNA in EHL and LW pigs were similar, which increased with age before day 120 and kept high level thereafter. The relative abundance of hepatic GHR mRNA was lower in male EHL than in male LW (P<0.01), but not than in female EHL pigs (P>0.05). (2) The changes of hepatic IGF-I mRNA abundance and the protein level of IGF-I in liver tissue with ages were consistent with that of hepatic GHR mRNA abundance. There was a strong correlation between GHR and IGF-I mRNA level (R=0.70, P<0.01, R=0.58 in LW, P<0.01; R=0.75 in EHL, PO.01), GHR mRNA and IGF-I protein level (R=0.67, P<0.01), IGF-I mRNA and protein level (R=0.78, P<0.01). The ABWG also correlated strongly with hepatic GHR mRNA (R=0.95, PO.01), IGF-I mRNA (R=0.91, PO.01) and IGF-I protein level (R=0.97, PO.01). The IGF-I mRNA (PO.01) and protein level (PO.05) were lower in male EHL pigs than male LW pigs, but not than in female EHL pigs (P>0.更多还原