【作者】 李鹏；

【导师】 王哲；

【作者基本信息】 吉林大学 ， 临床兽医学， 2012， 博士

【摘要】 本研究利用荧光定量PCR和ELISA等分子生物学技术以及犊牛肝细胞体外原代培养方法，检测了酮病奶牛肝脏脂肪酸氧化代谢关键酶表达水平变化规律，同时观察了NEFA、BHBA、葡萄糖等代谢产物和胰岛素、胰高血糖素、瘦蛋白等内分泌因子对体外培养的犊牛肝细胞脂肪酸氧化代谢关键酶表达水平的影响。证实了酮病奶牛肝脏脂肪酸氧化代谢途径关键酶类表达水平降低，同时探讨了各种代谢产物和内分泌因子对其关键酶表达水平的调控作用。为进一步揭示奶牛酮病的发生机理和制定相应的防治措施奠定了理论基础和实践依据。建立了脂肪酸氧化代谢途径关键酶类基因表达荧光定量PCR检测方法。根据GenBank中发表的牛ACSL、CPTⅠ、CPTⅡ、ACADL、HMGCS、ACC等基因序列，设计PCR引物，分别扩增出用于基因表达水平定量的目的片段，成功建立了各基因的荧光定量PCR检测方法。检测了酮病奶牛肝脏脂肪酸氧化代谢途径关键酶类基因表达水平。与健康奶牛相比，酮病奶牛肝脏组织中ACSL mRNA和蛋白表达水平显著增高，而CPTⅠ、CPTⅡ、ACADL、HMGCS、ACC mRNA和蛋白表达显著降低；实验奶牛血清NEFA浓度与肝组织中ACSL蛋白含量呈显著正相关，而与CPTⅡ、ACADL、HMGCS、ACC蛋白含量呈显著负相关；血清BHBA浓度与肝组织中与CPTⅡ、ACADL、HMGCS蛋白含量呈显著负相关。表明酮病奶牛肝脏脂肪酸氧化代谢能力减弱，血清中高水平的NEFA和/或BHBA是引起酮病奶牛肝脏脂肪酸氧化代谢能力减弱的关键因素。评价了代谢产物对肝细胞脂肪酸氧化代谢关键酶表达的影响。首先通过向培养液中添加不同浓度的外源性NEFA（0、0.2、0.4、0.8、1.6、3.2mM）、BHBA（0、0.5、0.75、1.0、1.5、3.0mM）、葡萄糖（0、1.0、2.0、4.0、8.0、16.0mM），观察代谢产物对肝细胞脂肪酸氧化代谢关键酶基因表达的影响。结果显示：1） NEFA显著促进犊牛肝细胞ACSL、ACADL、HMGCS的转录和翻译，呈剂量依赖性；显著抑制ACC的转录和翻译；对CPTⅠ的转录和翻译呈现低浓度促进而高浓度抑制的双重调控作用；但并未对CPTⅡ表现出明显的调控作用。2）BHBA显著促进犊牛肝细胞中ACSL的转录和翻译，呈剂量依赖性；显著抑制犊牛肝细胞中CPTⅠ、HMGCS的转录和翻译，呈剂量依赖性；但并未对CPTⅡ、ACADL、ACC等基因表现出显著的调控作用。3）GLU对ACSL的转录和翻译呈现低浓度促进而高浓度抑制的双重调控作用；显著抑制CPTⅡ、HMGCS的转录和翻译；显著促进ACC的转录和翻译，呈剂量依赖性；GLU可以促进CPTⅠ转录，但对其翻译水平没有明显的调控作用；GLU并未对ACADL的转录和翻译产生显著的调控作用。说明NEFA、BHBA、葡萄糖等代谢产物参与肝脏脂肪酸氧化代谢过程的调节。考察了内分泌因子对肝细胞脂肪酸氧化代谢关键酶表达的影响。首先通过向培养液中添加不同浓度的外源性内分泌因子胰岛素（0、5、10、20、50、100nM）、胰高血糖素（0、0.01、0.1、1、10、100nM）、瘦蛋白（0、2.5、5、10、50、100ng/mL），观察内分泌因子对肝细胞脂肪酸氧化代谢关键酶基因表达的影响。结果显示：1） INS显著抑制肝细胞ACSL、CPTⅠ、ACADL基因的转录和翻译，呈剂量依赖性；显著促进ACC基因的转录和翻译，呈剂量依赖性；显著抑制CPTⅡ基因转录，但对其翻译水平没有明显的调控作用；对HMGCS基因未表现出明显的调控作用。2）GLN显著提高ACSL、CPTⅠ基因的转录和翻译，显著抑制HMGCS、ACC基因的转录和翻译，呈剂量依赖性；GLN可以显著促进CPTⅡ基因转录，但并未对其翻译水平产生明显影响；GLN对ACADL基因未产生明显的调控作用。3）LP显著促进肝细胞ACSL、CPTⅠ、CPTⅡ、ACADL基因的转录和翻译，呈剂量依赖性；显著抑制ACC基因的转录和翻译，呈剂量依赖性；但对HMGCS基因未产生明显的调控作用。指示胰岛素抑制而胰高血糖素、瘦蛋白可促进肝脏脂肪酸氧化代谢过程。通过以上实验得出如下结论：1）酮病奶牛肝脏脂肪酸氧化代谢能力减弱，血液中高水平的NEFA和/或BHBA是抑制肝脏脂肪酸氧化代谢的关键调控因素。2）酮病奶牛肝脏脂肪酸活化增强而氧化代谢能力减弱，可能导致大量活化的脂酰辅酶A趋向于再酯化生成甘油三酯，从而引发奶牛肝脂沉积。3）葡萄糖、NEFA和BHBA等代谢产物和胰岛素、胰高血糖素和瘦蛋白等内分泌因子可以通过调节脂肪酸氧化代谢关键酶基因的转录和翻译影响肝脏脂肪酸氧化代谢过程。更多还原

【Abstract】 An underlying mechanism on fatty acid oxidation metabolism capability in theliver of ketotic cows will be brought to light through analyzing the expressing level ofkey enzymes such as acyl-CoA synthetase long-chain (ACSL), carnitinepalmitoyltransferase Ⅰ (CPTⅠ), carnitine palmitoyltransferase Ⅱ (CPTⅡ), acyl-CoAdehydrogenase long-chain (ACADL),3-hydroxy-3-methylglutaryl-CoA synthase(HMGCS), and acetyl-CoA (ACC) in the liver of ketotic cows, and the effect ofmetabolite and hormone on those key enzymes mRNA and protein levels in hepatocytesin vitro. Ⅰn this study, cytobiology and molecular biology were applied to research thealterations of fatty acid oxidation capability in the liver of ketotic cows. The results areas follow:Compared with non-ketotic cows, mRNA abundance of ACSL was increased by5.7-fold in ketotic cows. However, mRNA abundance of CPT Ⅰ, CPT Ⅱ, ACADL,HMGCS, and ACC were decreased by18%,70%,44%,52%, and64%, respectively,compared with non-ketotic cows. ACSL protein levels in the liver of ketotic cows weresignificantly higher, whereas levels of the following enzymes were significantly lower:CPT Ⅱ, ACADL, HMGCS, and ACC. Serum glucose concentration did not appear to beassociated with protein levels of ACSL, CPT Ⅰ, CPT Ⅱ, ACADL HMGCS, or ACC inthe liver of experimental cows. However, serum NEFA concentration was negativelycorrelated with protein levels of CPT Ⅱ, HMGCS, ACADL, and ACC and waspositively correlated with ACSL in experimental cows. Serum BHBA concentrationwas negatively correlated with protein levels of CPT Ⅱ, HMGCS, and ACADL inexperimental cows.Bovine hepatocyte was cultured by the modified two collagenase perfusion. Theeffects of NEFA (0、0.2、0.4、0.8、1.6、3.2mM), BHBA (0、0.5、0.75、1.0、1.5、3.0mM), and glucose (0、1.0、2.0、4.0、8.0、16.0mM) on mRNA andprotein expression of ACSL, CPTⅠ, CPTⅡ, ACADL, HMGCS, ACC in hepatocyte were detected by real-time PCR and ELISA. The results showed that: with increasedconcentration of NEFA, ACSL, ACADL, HMGCS transcription and translation levelswere increased, and ACC transcription and translation levels were decreased in culturedbovine hepatocytes, transcription was decreased with NEFA but translation levels wasnot changed, the transcription and translation of CPTⅠwere increased with lowerconcentration of NEFA and then decreased with increasing concentration of NEFA inthe media, and there was no obvious change in the CPTⅡtranscription and translation.With increased concentration of BHBA, ACSL transcription and translation levels wereincreased, and CPTⅠ, HMGCS transcription and translation were decreased in culturedbovine hepatocytes. There was no obvious change in the CPTⅡ, ACADL, ACCtranscription and translation levels with increasing BHBA concentrations in the culturemedia of bovine hepatocytes. With increased concentration of glucose, ACCtranscription and translation levels were increased, and CPTⅡ, HMGCS transcriptionand translation were decreased in cultured bovine hepatocytes, CPTⅡ transcriptionlevels were increased with glucose but translation levels was not changed. Thetranscription and translation of ACSL were increased with lower concentration ofglucose and then decreased with increasing concentration of glucose in the media. Therewas no obvious change in the ACADL transcription and translation levels withincreasing glucose concentrations.Bovine hepatocyte was cultured by the modified two collagenase perfusion. Theeffects of insulin (0、5、10、20、50、100nM), glucagon (0、0.01、0.1、1、10、100nM), and leptin (0、2.5、5、10、50、100ng/mL) on mRNA and proteinexpression of ACSL, CPTⅠ, CPTⅡ, ACADL, HMGCS, ACC in hepatocyte weredetected by real-time PCR and ELISA. The results showed that: with increasedconcentration of insulin, ACC transcription and translation levels were increased, andACSL, CPTⅠ, ACADL transcription and translation levels were decreased in culturedbovine hepatocytes, CPTⅡ transcription was decreased with insulin but translationlevels was not changed, and there was no obvious change in the HMGCS transcriptionand translation. With increased concentration of glucagon, ACSL, CPTⅠ transcriptionand translation levels were increased, and HMGCS, ACC transcription and translationlevels were decreased in cultured bovine hepatocytes, CPTⅡ transcription was increased with glucagon but translation levels was not changed, and there was noobvious change in the ACADL transcription and translation.With increasedconcentration of leptin, ACSL, CPTⅠ, CPTⅡ, ACADL transcription and translationlevels were increased, and ACC transcription and translation levels were decreased incultured bovine hepatocytes, and there was no obvious change in the HMGCStranscription and translation.As above-mentioned, fatty acid β-oxidation capability was altered in the liver ofketotic cows. Increased activation and decreased β-oxidation of fatty acid suggests morefatty acyl-CoA esterification into triacylglyceride, which may lead to liver lipidosis.High serum NEFA and BHBA concentrations play key roles in metabolism of fatty acidin ketotic cows. The intermediate metabolic products such as NEFA, BHBA, glucoseand hormeones such as insulin, glucagon, leptin could regulate the fatty acid oxidationmetablism capability through promoting or inhibiting the expression of fatty acidoxidation-related enzymes.更多还原