【作者】 张艳芳；

【导师】 汪以真；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 本研究以肉脂型的金华猪和瘦肉型的长白猪为试验对象,在比较研究两猪种胴体品质和肉质的基础上,探讨脂肪酸β-氧化关键酶肉碱棕榈酰转移酶1(CPT1)在金华猪和长白猪不同组织中的表达特异性、不同生长阶段的表达规律及品种间的差异,研究CPT1基因表达在脂肪沉积中的作用,及其与体脂和肌内脂肪沉积的关系。通过猪肌内前体脂肪细胞体外培养模型的建立,利用CB1激动剂△9-四氢大麻酚(△9-THC)和抑制剂利莫那班(SR141716)探讨大麻素1型受体(CB1)基因对CPT1调控的分子机制。主要研究结果如下：1.金华猪、长白猪胴体性状及肉质性状的比较研究结果表明,180日龄金华猪瘦肉率和眼肌面积均显著小于同日龄的长白猪(p<0.01);金华猪肌内脂肪含量显著高于长白猪(p<0.01),金华猪背膘厚、脂肪率和皮脂率均显著高于长白猪(p<0.01);金华猪压出水比显著低于长白猪(p<0.01)；金华猪滴水损失低于长白猪,但差异不显著(p>0.05);金华猪和长白猪宰后45min肌肉pH值差异不显著(p>0.05),且均在正常范围内；肉色中金华猪L值显著高于长白猪(p<0.01),两猪种a值和b值差异不显著(p>0.05)。试验表明,金华猪和长白猪在胴体品质和肉质上存在显著差异,在肉质上,金华猪肉质优于长白猪。2.金华猪、长白猪血液生化指标及脂肪代谢相关酶活的比较研究与长白猪相比,金华猪具有较高的血清总胆固醇(TC)含量、低密度脂蛋白(LDL)和载脂蛋白B(Apo B)含量；具有较低的血清甘油三酯(TG)含量和脂蛋白a(Lp a)含量。在皮脂中,金华猪激素敏感脂肪酶(HSL)的活性显著低于长白猪(p<0.05),肝脂酶(HL)的活性显著高于长白猪(p<0.05)。在同一猪种上,HSL、脂蛋白酯酶(LPL)和HL的活性在脂肪组织中最高,肝脏组织中其次,肌肉组织中最低。3.猪CPTl基因表达的组织特异性、表达规律及品种差异研究CPT1基因的两种亚型L-CPT1和M-CPT1在猪的不同组织均有广泛的表达。L-CPT1 mRNA在肝脏、胰脏和十二指肠中表达量较高,在背最长肌、皮脂、脾和脑中表达量较低。M-CPT1 mRNA在背最长肌和皮脂中表达量较高,在肾脏、脾脏、脑、肺和十二指肠中表达量较低。在不同生长阶段的金华猪和长白猪上,L-CPT1和M-CPT1具有相似的表达规律,两基因的mRNA表达量都呈现先上升后下降的趋势,在30日龄时表达量达到最高。品种差异性比较研究的结果表明,在背最长肌中,金华猪的CPT1 mRNA表达量和蛋白表达量均显著高于长白猪(p<0.01),且与肌内脂肪含量呈正相关；在皮下脂肪和腹脂中,金华猪的CPT1 mRNA表达量和蛋白表达量均显著低于长白猪(p<0.01),且与体脂肪率呈负相关。提示CPT1基因与体脂和肌内脂肪沉积有密切的关系。4.CB1对CPT1基因表达和脂肪沉积的影响通过猪肌内前体脂肪细胞体外培养模型的建立,利用CB1激动剂A9-THC和抑制剂SR141716研究CB1对脂肪酸p-氧化关键酶CPT1的调控作用及对脂肪沉积的影响。研究结果表明,CB1激动剂激活CB1基因表达后,显著降低了CPT1基因的表达,同时增加了细胞内脂滴的沉积,减少了培养基中甘油的含量。CB1抑制剂SR141716抑制CB1基因的表达后,显著提高了CPT1基因的表达,同时减少了细胞内脂滴的沉积,增加了培养基中甘油的含量。在培养基中添加CPT1的抑制剂乙莫克舍(ETM)后,未对CB1基因的表达产生影响,推测CB1可能是CPT1的上游基因。A9-THC和ETM共同处理猪肌内脂肪细胞后,二者产生正协同作用,显著降低了CPT1基因的表达,增加了细胞内脂滴的沉积,减少了培养基中甘油的含量。同时研究还发现,A9-THC下调CPT1基因的表达时,伴随着PPARa基因的表达降低；SR141716上调CPT1基因的表达时,也伴随着PPARa基因的表达升高。上述结果提示,CB1可通过调控CPT1基因的表达进而影响脂肪沉积。在此过程中,PPARα信号通路可能发挥着重要的作用。更多还原

【Abstract】 This study was conducted to compare the carcass quality and meat quality between Jinhua pigs and Landrace pigs. Based on these results, detected the tissue-specific, developmental expression pattern and the breed differences of CPT1, and analyzed its relationship with intramuscular fat and body fat deposition. This study established a pig intramuscular preadipocytes culture model successfully. Use CB1 agonist (△9-THC) and inhibitors (SR141716) to explore the molecular regulation mechanism of CPT1 and the effects on fat deposition in vitro. Results are as bellow:1. Comparative study of carcass quality and meat quality between Jinhua pigs and Landrace pigsOn age of 180 days, slaughter weight of Jinhua pigs was significantly lower than the Landraces (p<0.01); lean percentage and loin eye area were also significantly lower than the Landraces (p<0.01); intramuscular fat content of Jinhua pigs was significantly higher than that of Landraces (p<0.01); back fat thickness, fat percentage, and skin fat percentage of Jinhua pigs were significantly higher than Landraces (p<0.01); water pressure of Jinhua pigs were significantly lower than Landraces (p<0.01); drip loss of Jinhua pigs lower than the Landraces, but the difference was not significant (p> 0.05); pH value of Jinhua pigs and Landrace have no significant difference (p> 0.05. L value of Jinhua pigs was significantly higher than Landraces (p<0.01), a value and b value of the two breeds had no differences (p>0.05). These results showed that Jinhua pigs and Landrace had great differences in meat quality; Jinhua pigs had better meat quality than Landraces.2. Comparative study of blood biochemical parameters and lipid metabolism related enzymes activates between Jinhua pigs and Landrace pigsCompared with Landrace, Jinhua pigs had higher serum total cholesterol (TC) content, low density lipoprotein (LDL) and apolipoprotein B (ApoB) levels, and had lower serum triglyceride (TG) content and lipoprotein a (Lpa). HSL activity of Jinhua pigs in subcutaneous adipose tissue was significantly lower than Landrace, and HL activity of Jinhua pigs in subcutaneous adipose tissue was significantly higher than Landrace. In the same breed of pig, HSL, LPL and HL activity were highest in adipose tissue, liver secondly, the lowest in muscle.3. The study on the tissue-specific, developmental expression pattern and breed differences of CPT1Tissue-specific study results showed that L-CPT1 and M-CPT1, the two subtypes of CPT1, were widely expressed in all tissues we studied. L-CPT1 mRNA was highly expressed in liver, pancreas and duodenum, and lower in Longissimus Muscle, subcutaneous adipose tissue, spleen and brain. M-CPT1 mRNA was highly expressed in Longissimus Muscle, subcutaneous adipose tissue, and lower in kidney, spleen, brain, lung and duodenum. The developmental expression pattern of different growth stage study results showed that both of L-CPT1 and M-CPT1 mRNA expression were increased before 30 days of age, then decreased; And 30 days of age had their highest expression level. The breed difference study results showed that in Longissimus Muscle, CPT1 gene expression and protein expression levels of Jinhua pigs were both significantly higher than the Landrace (p<0.01), and positively correlated with the IMF. In subcutaneous adipose tissue, CPT1 gene expression and protein expression levels of Jinhua pigs were both significantly lower than the Landrace (p<0.01), and negatively correlated with fat percentage. These results indicated that CPT1 plays an important role in fat deposition and IMF deposition.4. Study of the molecular mechanism of CB1 regulation on CPT1 and the relationship to fat depositionIn vitro, culture system of porcine intramuscular adipocytes was successfully established. Use the CB1 agonist (△9-THC) and inhibitors (SR141716) to explore the molecular regulation mechanism of CPT1 and the effect on the fat deposition. The results showed that△9-THC significantly increased CB1 mRNA levels, significantly decreased CPT1 mRNA levels, increased the lipid accumulation of porcine adipocytes, and reduced the medium glycerol content. SR141716 significantly decreased CB1 mRNA levels, significantly increased CPT1 mRNA levels, decreased the lipid accumulation of porcine adipocytes, and increase the medium glycerol content. Treat the porcine adipocytes with CPT1 inhibitor ETM did not affect the CB1 gene expression, suggesting that CB1 may be the upstream gene of CPT1. Treated the porcine adipocytes with△9-THC and ETM further proved that can increased the lipid accumulation and reduced the medium glycerol content. At the same time, we also found that inhibited the gene expression of CPT1 by△9-THC significantly inhibited the PPARa gene expression; activated the CPT1 gene expression by SR141716 significantly enhanced the gene expression of PPARa. These results revealed that CB1 regulate the gene expression of CPT1, and then affect the fat deposition, and PPARa signal pathway may play an important role in this process.更多还原