【作者】 邓俊良；

【导师】 崔恒敏； 王哲；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 博士

【摘要】 以围产期能量代谢障碍为病理学基础的酮病和脂肪肝是奶牛重要的群发性常见多发病。研究表明,酮病、脂肪肝是奶牛能量负平衡的结果,脂肪动员、肝脂沉积、低糖血症和酮体生成增多是奶牛酮病和脂肪肝的主要环节。围产期奶牛能量代谢特点是干物质摄入减少及能量负平衡,糖异生是反刍兽生糖的主要途径,磷酸烯醇式丙酮酸羧激酶(PEPCK)是糖异生途径的主要关键酶,通过上调PEPCK基因的表达量可直接影响肝脏糖异生的能力,从而缓解低血糖。脂肪动员是缓解能量负平衡的唯一途径。一方面弥补糖异生作用降低所引起的能量亏欠;另一方面释放大量游离脂肪酸(NEFA)进入血液及肝脏,可引发脂肪肝和酮病。而脂肪动员是由甘油三酯脂肪酶(又称激素敏感脂肪酶HSL)催化完成的。HSL的表达量直接体现脂肪动员的程度,所以调控脂肪动员是防治脂肪肝、酮病等围产期能量代谢障碍性疾病的重要措施。肝脂沉积也是脂肪肝、酮病发病根源之一。在肝脂沉积过程中,硬脂酰CoA去饱和酶(SCD)是单不饱和脂肪酸(MUFA)生物合成的限速酶,SCD可促进甘油三酯(TG)合成底物的形成,从而促进TG的合成。所以调控SCD酶的表达能直接调控脂肪的合成。而神经内分泌因子和代谢产物是调节糖代谢、脂肪代谢,缓解酮病和脂肪肝的主要途径。因此本研究将通过体外(肝细胞和脂肪细胞培养)途径,运用分子生物学技术(定量RT-PCR、荧光定量PCR法),重点研究部分神经内分泌因子及代谢中间产物对奶牛糖异生关键酶(PEPCK)、甘油三酯合成关键酶(SCD)基因表达和脂肪动员关键酶(HSL)的活性及基因表达的分子调控机制。阐明神经内分泌因子及代谢中间产物在奶牛酮病、甘油三酯合成、脂肪动员中的调控作用,为从基因水平和蛋白质水平揭示奶牛酮病、脂肪肝发病机制奠定分子生物学基础和理论依据。实验选取临床检查健康的新生荷斯坦犊牛,按照本实验建立的犊牛肝细胞原代单层培养方法进行培养,观察其生长形态,肝细胞培养至48h完全贴壁,为加样的最佳时机。以β-actin为内参,以相同的cDNA为模板,优化了RT-PCR反映条件,成功地对目的基因(PEPCK、β-actin)进行了克隆扩增、克隆、鉴定及测序,测序结果与GeneBank报道一致。在肝细胞培养到48h时,分别在培养液中添加0、1、10、100和1000nmol/mL胰岛素(INS),0、1、10、100和500pg/mL胰高血糖素(GN),0、5、10、15、20、30、40、50、60、100和150ng/mL胰岛素样生长因子-Ⅰ(IGF-Ⅰ),每浓度梯度设三个重复。通过RT-PCR法检测INS、GN和IGF-Ⅰ对糖异生关键酶PEPCKmRNA表达的影响。采用SYBR greenⅠ染料法建立了适时荧光定量PCR的优化反应条件,成功地对目的基因(β-actin、SCD)进行了扩增,两基因的扩增效率相同。在肝细胞培养到48h时,分别在培养液中添加0、5、10、20、50IU/mL的INS,0、50、100、500、1000pg/mL的GN,0、50、100、500、1000pg/mL神经肽Y(NPY),每浓度梯度设三个重复,以β-actin为内参基因,用荧光PCR方法检测肝细胞SCDmRNA的表达。通过犊牛前脂肪细胞原代单层培养(整个培养期为14d),培养至第8、12d,用油红0工作液和台盼蓝工作液对脂肪细胞分别进行染色,观察其生长形态;第14d,脂肪细胞分化成大的单脂滴的脂肪细胞。此时,在生长良好的脂肪细胞培养介质中分别添加0、10、20、30、40、50μg/L的IGF-Ⅰ,0、100、250、500、750、1000nmol/L的胰高血糖素样肽Ⅰ(GLP-Ⅰ),0、10、20、40、80、160mg/L的地塞米松、0、0.5、1.0、1.5、2.0、2.5mmol/L的油酸,0、10、20、30、40、50mg/L的乳酸(每浓度梯度设三个重复),再分别进行培养24h后提取细胞总RNA,通过荧光定量PER扩增,观察IGF-Ⅰ、GLP-Ⅰ、地塞米松、油酸及乳酸处理的脂肪细胞HSLmRNA丰度的变化。提取处理的脂肪细胞总蛋白,测定所提细胞总蛋白浓度,用脂肪酶测定试剂盒测定处理脂肪细胞HSL活性的变化。结果显示:1.INS和IGF-Ⅰ能显著下调体外培养犊牛肝细胞PEPCK基因表达,并呈现剂量依赖性,但INS浓度在1～10和100～1000nmol/mL时抑制效应趋缓;IGF-Ⅰ浓度在10～15ng/mL时抑制效果稍趋平缓外,其抑制作用有明显剂量依赖性,IGF-Ⅰ＞100ng/mL后几乎检测不到PEPCK。GN能促进犊牛肝细胞PEPCK基因mRNA的表达,具有浓度依耐性,各浓度组间差异极显著,在10～100pg/mL时促进效果最明显,浓度≥100pg/mL上调效应趋于饱和。因此,INS和IGF-Ⅰ通过减弱而GN通过促进肝PEPCKmRNA表达来调节肝糖异生。2.INS浓度≥5IU/mL、NPY浓度≥50pg/mL对体外培养的犊牛肝细胞SCDmRNA表达有明显促进肝脏,且呈剂量依赖促进效应。GN浓度≥50pg/mL明显抑制肝脏SCDmRNA表达,且呈剂量依赖抑制效应。由此表明,INS和NPY可通过促进SCDmRNA表达,促进肝脂沉积:而GN可通过抑制SCDmRNA表达,减少肝脂沉积。3.IGF-Ⅰ、GLP-Ⅰ、油酸、乳酸对体外培养脂肪细胞HSLmRNA表达及酶活性均呈现明显抑制作用,并存在剂量依赖性。IGF-Ⅰ浓度≥20μg/L对HSLmRNA的表达抑制作用显著,浓度≥30μg/L对酶活性抑制作用显著;GLP-Ⅰ浓度≥100nmol/L对HSLmRNA表达及酶活性抑制作用显著:油酸浓度≥1.0mmol/L显著抑制HSLmRNA表达,浓度≥0.5mmol/L极显著抑制HSL活性;乳酸浓度≥20mg/L显著抑制HSLmRNA表达,浓度≥40mg/L显著抑制HSL活性。地塞米松浓度≥20mg/L显著促进HSLmRNA表达,浓度≥10mg/L显著促进HSL活性。表明IGF-Ⅰ、GLP-Ⅰ、乳酸、油酸、地塞米松可通过影响奶牛脂肪细胞内HSLmRNA表达和酶活性影响脂肪代谢,除地塞米松可促进脂肪分解外,GF-Ⅰ、GLP-Ⅰ、乳酸、油酸均抑制脂肪分解,促进脂肪的沉积。更多还原

【Abstract】 The ketosis and adiposis hepatica, which based on the pathobiology agent of perinatal stage energy metabolism disturbance are significant cow frequently encountered diseases. Research indicates that ketosis and adiposis hepatica are caused by negative balance of energy and fat mobilization, hepar lipidosis, hypoglycemia. ketoplasia are the main element.The energy metabolism feature of cow in perinatal stage is decrease in dry matter englobement and negative balance of energy, glyconeogenesis is the main path of produceing glucose in ruminant, PEPCK is the key enzyme of glyconeogenesis, glyconeogenesis and hypoglycemia can be effected through promoting PEPCK gene expression.Fat mobilization is the only path to relievenegative balance of energy,which can retrieve negative balance of energy as well as produce massive free fatty acid to initiate ketosis and adiposis hepatica. Fat mobilization is catalysised by HSL. So the expression of HSL personalizes mobilization level directly and controlling fat mobilization is significant measure to prevent and cure perinatal stage energy metabolism disturbance.Hepar lipidosis is another invasion origin of ketosis and adiposis hepatica.SCD is rate-limiting enzyme of MUFA production, which can promote triglyceride synthesis. So. fat synthesis can be controlled by controlling SCD expression.Neuroendocrine agent and metabolic are main path to control glycometabolism, fat metabolism and relieve ketosis and adiposis hepatica. The purpose of this research is to investigate molecule control mechanism of PEPCK,SCD,HSL expression and HSL activity, illuminate regulation of neuroendocrine agent and metabolic in the course of ketosis. triglyceride production and fat mobilization, establish molecular biological theory fundament for revealling pathogenesy in the genic and proteinaceous level.Health newborn calves were selected for primary monolayer hepatocytes culture in vitro, which was the best time for spotting while adherent cells emerged after 48h.Target genes(β-actin,PEPCK) were amplificated, cloned, identified and sequenced successfully by applying the reference ofβ-actin, the template of same cDNA. optimized reflect condion of RT-PCR. The results were at equal with Genebank’s. INS of 0, 1, 10, 100, 1000nmol/mL and GN of 0, 1, 10, 100, 500pg/mL and IGF-I of 0, 5. 10, 15, 20, 30. 40, 50, 60, 100, 150ng/mL (three array in each gradient) were added to culture fluid.The effect of INS, GN, IGF-Ⅰon expression of PEPCKmRNA were detected by RT-PCR method.Target genes(P-actin,SCD) were successfully amplificated through SYBR greenⅠreal-time quantitative PCR and coined. INS of 0, 5, 10, 20, 50IU/mL and GN of 0, 50, 100, 500, 1000pg/mL, and NPY of 0, 50, 100, 500, 1000pg/mL(three array in each gradient) were added to culture fluid. Then the expression of SCD mRNA was detected applying the reference ofβ-actin and fluorescent PCR methods to detect the expression of SCD mRNA.Primary monolayer preadipocyte was cultured for 14d. Observe cells at 8d and 12d after staining with oil red O and trypan blue solution. IGF-1 of 0, 10. 20, 30, 40, 50ug/L. GLP-Ⅰof 0, 100, 250, 500, 750, 1000nmol/L, dexameth of 0, 10, 20, 40, 80,160mg/L, oleic acid of 0, 0.5, 1.0, 1.5, 2.0, 2.5mmol/L, and lactic acid of 0, 10, 20. 30, 40, 50mg/L were added to culture fluid after simple lipids emerged at 14d, then isolated total RNA after 24h. The expression of HSL mRNA was detected by fluorescent quantitation PCR in adipocyte handled with IGF-Ⅰ,GLP-Ⅰ, dexameth. oleic acid and lactic acid. Then total protein was isolated and detected. HSL activity was detected using lipase kit.The results were as follows:1. The PEPCK mRNA expression was notably inhibited by INS and IGF-Ⅰ. and the effect was dose dependent. While the concentration of INS was between 1 and 10nmol/mL or between 100 and 1000nmol/mL.the suppression was gently. The suppression of IGF-Ⅰwas dose dependent. While the concentration of IGF-Ⅰwas between 10 and 15ng/mL, the suppression was gently, and while the concentration was more than 100ng/mL, there was nearly no PEPCK. The PEPCK mRNA expression dose-dependently increased by GN. Liver gluconeogenesis ability was adjusted by INS and IGF-Ⅰthrough inhititing PEPCK mRNA expression and adjusted by GN through increasing PEPCK mRNA expression.2. The expression of SCD mRNA was notably promoted in hepatocytes while the concentration of INS and NPY was more than 5IU/mL and 50pg/mL respectively. This expression was dose dependent. The expression was notably suppressed while the concentration of GN was more than 50pg/mL. This suppression was dose dependent. INS and NPY promoted SCD mRNA expression and liver fat storing. GN inhibited SCDmRNA express and liver fat storing.3. The expression of HSL mRNA and the activity of HSL were suppressed in adipose cell, treated with IGF-Ⅰ, GLP-Ⅰ, oleic acid and lactic acid. This effecion was dose dependent. While the concentration of IGF-Ⅰ, GLP-Ⅰ, oleic acid, lactic acid was more than 20ng/L,1.0mmol/L and 20mg/L respectively, the suppression towards HSL mRNA abundance was notable. While the concentration was more than 30ug/L,0.5mmol/L and 40mg/L respectively, the suppression towards HSL activity was notable. While the concentration of GLP-Ⅰwas more than 100nmol/L, the suppression towards HSL mRNA abundance and HSL activity was notable. While the concentration of dexameth was more than 20mg/L, the expression of HSL mRNA was promoted, and while the concentration was more than 1 0mg/L, the HSL activity was promoted. It was concluded that IGF-Ⅰ, GLP-Ⅰ, oleic acid, lactic acid, dexameth regulated the fat lipoclasis through regulating the abundance of HSL mRNA and the HSL activity. Dexameth promoted lipoclasis and IGF-Ⅰ, GLP-Ⅰ, oleic acid, lactic acid inhibited lipoclasis, and then promote fat storing.更多还原