【作者】 闫峻；

【导师】 孙超；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2013， 博士

【摘要】 本研究通过活体药物处理、构建超表达及干扰载体并处理鸡原代脂肪细胞、球状脂联素处理细胞等技术手段,研究脂联素对肉鸡活体脂肪沉积、肌肉品质和鸡脂肪细胞中脂代谢的影响及其调控机制。试验获得以下结果：1.为了研究肉鸡脂联素表达变化对活体脂肪沉积及肌肉品质的影响,试验采用罗格列酮及地塞米松处理肉鸡,连续处理三周,检测血清中脂联素水平,脂联素及其受体基因的表达规律,同时研究肉鸡体脂沉积,检测脂肪组织中脂代谢关键基因的表达。结果表明：(1)罗格列酮显著上调肉鸡血清中脂联素水平,地塞米松显著抑制血清中脂联索水平,并呈时间依赖性；(2)罗格列酮显著上调腹脂、皮脂、胸肌、腿肌和肝脏中脂联素及其受体AdipoR1基因的表达；(3)罗格列酮显著降低血清中TG和LDL-C水平,上调HDL-C水平,地塞米松显著上调血清中TG和LDL-C水平,下调HDL-C水平；(4)罗格列酮显著抑制腹脂和颈部皮脂以及胸肌和腿肌中的脂质沉积,而地塞米松显著增加肉鸡腹脂、腹部皮脂、颈部皮脂和腿部皮脂以及胸肌和腿肌中脂质沉积；(5)罗格列酮显著下调胸肌和腿肌中总FA、SFA和MUFA的含量,地塞米松则显著促进肌肉中脂肪酸的沉积；(6)罗格列酮处理组肌肉剪切力上升,滴水损失上升,肉色下降,而地塞米松组则相反。(7)罗格列酮显著上调脂肪、肝脏和胸肌中ATGL基因的表达,显著抑制脂肪及肝脏组织中CEBPα和FAS的表达。地塞米松显著上调CEBPα和FAS的表达,而显著抑制ATGL基因的表达。2.为了研究全长脂联素表达变化对鸡脂肪细胞分化的影响,试验构建了鸡脂联素基因超表达及干扰真核质粒载体,处理鸡原代脂肪细胞细胞。结果表明：(1)超表达脂联素抑制脂肪细胞分化及脂滴生成,而沉默脂联素基因则促进鸡脂肪细胞分化和脂滴生成；(2)超表达脂联素抑制了CEBPα和FAS的表达水平,而促进了ATGL的表达。干扰脂联素基因则促进了CEBPα和FAS的表达,抑制了ATGL的表达；(3)超表达脂联素激活了p38MAPK/ATF-2信号通路,SB253580能够阻断这种活化作用,而干扰脂联素则抑制了p38MAPK/ATF-2通路;(4)超表达脂联素抑制了TOR/p70S6Kinase信号通路,雷帕霉素则能够加强这种抑制作用,而干扰脂联素则激活了TOR/p70S6Kinase信号通路。3.为了研究全长脂联素的水解产物-球状脂联素对鸡脂肪细胞分化鸡脂质蓄积的影响,试验采用200μg/mL人重组球状脂联素处理鸡原代脂肪细胞。结果表明：(1)球状脂联素能够促进鸡脂肪细胞内源脂联素及AdipoR1基因表达；(2)球状脂联素抑制了鸡脂肪细胞分化和脂质沉积(3)球状脂联素上调CEBPα和FAS的表达,抑制了FAS表达；(4)球状脂联素上调p38MAPK/ATF-2蛋白磷酸化水平,并且能够修复SB253580介导的p38MAPK/ATF-2通路抑制作用；(5)球状脂联素抑制TOR/p70S6Kinase蛋白磷酸化水平,并且能够协同雷帕霉素进一步降低TOR/p70S6Kinase蛋白磷酸化。4.为了研究鸡脂联素对脂肪细胞中线粒体发育及功能的影响,我们构建了鸡脂联素腺病毒载体处理鸡原代脂肪细胞,并采用干扰真核载体处理细胞。结果表明：(1)超表达脂联素负调控鸡脂肪细胞分化和脂质沉积,超表达脂联素促进CEBPα、LPL和ATGL的表达,抑制FAS、A-FABP和ACC的表达；(2)超表达脂联素促进鸡脂肪细胞24h葡萄糖摄取和培养基中FFA的释放,而干扰脂联素则抑制脂肪细胞24h葡萄糖摄取和培养基中FFA的释放；(3)脂联素促进脂肪细胞中线粒体发育及线粒体内糖脂代谢功能基因的表达；(4)脂联素上调脂肪细胞线粒体膜电位；(5)超表达脂联素促进p38MAPK和ERK的磷酸化水平,抑制TOR蛋白磷酸化。综上所述,本研究证明了罗格列酮和地塞米松分别上调或下调肉鸡活体内脂联素的表达,并且脂联素表达的变化与肉鸡体脂沉积以及肌肉品质成负相关关系；超表达脂联素以及重组球状脂联素显著抑制了脂肪细胞分化,而干扰脂联素基因则显著促进脂肪细胞分化,p38MAPK和TOR通路参与脂联素对鸡脂肪细胞分化的调节；超表达脂联素促进了鸡脂肪细胞中线粒体的发育并激活线粒体中糖脂代谢功能,并且上调了线粒体膜电位；ERK和TOR信号通路参与了脂联素对线粒体功能的调节。更多还原

【Abstract】 The present study was conducted to investigate the effect of adiponectin on fat deposition and muscle quality of broiler chicken in vivo and lipid metabolism of chicken adipocyte in vitro, mean while, the mechanism of the action of adiponectin was also observed. Rosiglitazone and dexamethasone were used to treat broilers in vivo for three weeks; over-expression and interference vectors targeting of chicken adiponectin gene were constructed and transfected into chicken preadipocytes;200μg/mL recombinant globular adiponectin was used to treat chicken preadipocyte. At last, recombinant adenovirus vector targeting chicken adiponectin was constructed and treated chicken preadipocytes to clarify the effect of adiponectin on mitochondria development and its function in chicken adipocyte. Results showed that:1.(1) Rosiglitazone increased serum adiponectin concentration in a time-dependent manner, while dexamethasone increased serum adiponectin;(2) Rosiglitazone up-regulated the expression levels of adiponectin and adipoRl in tissues, while dexamethasone decreased adiponectin and adipoRl mRNA levels;(3) Rosiglitazone decreased the levels of serum lipids, while dexamethasone increased serum lipids;(4) Rosiglitazone decreased abdominal fat, neck subcutaneous fat deposition and lipids in muscle, while dexamethasone increased abdominal fat, neck subcutaneous fat, leg subcutaneous fat, abdominal subcutaneous fat and lipids in muscle;(5) Rosiglitazone down-regulated total FA, SFA and MUFA in muscle, while dexamethasone increased the deposition of total FA,SFA and MUFA in muscle;(6) Rosiglitazone increased the expression of ATGL in tissues and inhibited CEBPa and FAS, while dexamethasone have the opposite effect.2.(1) overexpression adiponectin inhibited the differentiation of chicken preadipocyte;(2) adiponectin decreased the expression of CEBPa and FAS, while increased ATGL expression;(3) over-expression of adiponectin could activate p38MAPK/ATF-2pathway and also repaired the suppression of p38MAPK and ATF-2activation by SB253580;(4) over-expression of adiponectin suppressed TOR/p70S6Kinase pathway in chicken adipocytes and may further reduce the phosphorylation level of the TOR and p70S6Kinase based on the inhibitory effect of TOR.3.(1) globular adiponectin inhibited the differentiation of chicken preadipocyte;(2) globular adiponectin decreased the expression of CEBPa and FAS, while increased ATGL expression;(3) globular adiponectin could activate p38MAPK/ATF-2pathway and also repaired the suppression of p38MAPK and ATF-2activation by SB253580;(4) globular adiponectin suppressed TOR/p70S6Kinase pathway in chicken adipocytes and may further reduce the phosphorylation level of the TOR and p70S6Kinase based on the inhibitory effect of TOR.4.(1) over-expression of adiponectin inhibited chicken adipocyte differentiation, increased the expression of CEBPa, LPL and ATGL, while decreased the expression of FAS, A-FABP and ACC;(2) over-expression of adiponectin promoted glucose uptake and FFA release in medium during24h;(3) over-expression of adiponectin promoted mitochondria development and increased the expression of glucose and lipid metabolism marker genes in the mitochondria;(4) over-expression of adiponectin increased mitochondrial membrane potential;(5) over-expression of adiponectin activated p38MAPK and ERK pathway, while inhibited the activation of TOR pathway.In summary, in our studies, we proved that rosiglitazone and dexamethasone could regulate the levels of serum adiponectin and analyzed the impact of serum adiponectin on fat deposition and meat quality. Over-expression of full length of adiponectin and globular adiponectin had the same impact on the inhibitory of chicken preadipocyte differentiation. Over-expression of adiponectin promoted mitochondria development and also stimulated glucose and lipid metabolism in mitochondria. p38MAPK, ERK and TOR signaling pathways involved in the regulation of adiponectin on mitochondrial function.更多还原