脂联素介导绵羊肌内、内脏和皮下脂肪细胞生脂的基因表达模式与分子调控机理研究

Adiponectin is the highest adipokine that secretes by adipocyte, which is also the only one that negatively correlated with obesity up until now. It plays an important role in energy supply, lipid metabolism and glucose homeostasis, as well as anti-atherogenic. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin and type 2 diabetes. Studies of adiponectin and its receptors are mainly concentrated in human being, mouse, swine and chichen. Very few reports of adiponectin in ruminant species could be found, especially in sheep. .The present study, focusing on the Lanzhou fat-tailed sheep, will first clone the full length cDNA encoding adiponectin (adpn) and its receptors (adpnR). Temporal and spatial specificity of genes expression will be carried out by using quantitative real-time PCR (qPCR). Correlation analysis between gene expression of adpn and adpnR and IFM will be implement. Recombinant proteins will be induced to express in prokaryotic vectors pET-28a (+)-adpn, pET-28a (+)-adpnRs and purification process will be applied using fast protein liquid chromatography. The intramuscular fat (IMF), subcutaneous (s.c.) fat and visceral (v.c.) fat will be isolated and their adipocytes will be cultured in vitro. Mammalian ORF expression vectors of pReceiver-EU-adpn and pReceiver-EU-adpnR, eukaryotic expression vectors of pcDNA3.0-adpn and pcDNA-adpR will be constructed and transfected to cultured adipocytes. The influences of lipogenesis on above mentioned adipocytes under different concentrations of recombinant proteins will be exerted and differential expression levels of lipid metabolism genes will be analyzed by qPCR. Using technology of RNA interference (RNAi) to slience the expression of Adpn and adpnRs in cultured fat cells, the differences of gene expression of peroxisome proliferator-activate receptor ɑ (PPARɑ), AMP-activated protein kinase (AMPK) and p38 mitogen protein kinase (p38MAPK), which play key rols during signal transduction of adpn and adpnR, will be processed. Meanwhile, the lipogenesis and expression of fat-metabolismin-related genes in adpn and adpnR silent adpocytes of IMF, s.c. fat and v.c. fat will be elucdated.. These findings would initally reveal the molecular mechanism of lipogenesis in different fat dpots so as to provide some theoretical bases for finally clarifying body fat deposition and improving meat quality in sheep, as well as applying to the sheep breeding practice.

脂联素是脂肪细胞所分泌的细胞因子中含量最高、的唯一与肥胖呈负相关的细胞因子，参与调节脂肪代谢、葡萄糖代谢、能量稳态调节及抵抗炎症反应等生理过程。脂联素及其受体基因研究主要集中在人、小鼠、猪、鸡上，绵羊脂联素及其受体的研究国内外还未见报道。本研究以兰州大尾羊为材料，首次克隆绵羊脂联素及其受体基因全长cDNA序列，qPCR研究脂联素及其受体基因时空表达规律；通过体外分离和培养绵羊肌内脂肪、皮下脂肪和内脏脂肪细胞，研究重组脂联素及其受体对不同脂肪细胞生脂及生脂相关基因表达的差异;通过siRNA技术沉默脂联素及其受体基因表达，探讨对脂联素及其受体表达信号通路关键基因PPARɑ、AMPK和p38MAPK的表达差异；分析脂联素及其受体与肌内脂肪沉积关系。初步揭示脂联素及其受体基因调控绵羊脂肪组织生脂的分子机理，为最终阐明绵羊体脂沉积和改善肉品质提供依据，也为肉羊育种中优良性状利用提供理论有益参考。

脂联素与肥胖呈负相关，参与脂肪代谢、能量稳态调节和葡萄糖代谢等生理过程。脂联素及其受体基因克隆及功能研究主要集中在人、小鼠上，绵羊上未见相关报道。本研究以兰州大尾羊为实验材料，应用RACE技术首次克隆出脂联素基因2 101 bp（KJ159213）、脂联素受体1基因2 035 bp（KJ159212）和脂联素受体2基因1 809 bp（KF921623）全长cDNA；研究了3种基因在绵羊肝、肾、心、肺、脾、皮下脂肪、内脏脂肪、肌肉、卵巢、睾丸、大肠、小肠、大脑、瘤胃和皱胃15种组织中的特异性表达规律，Adpn肌肉组织中表达最高，心脏和皱胃表达量次之；AdipoR1在皮下脂肪组织和内脏脂肪组织表达量最高，其次是脾脏、心脏、小肠；AdipoR2在皮下脂肪组织和内脏脂肪组织表达量最高，在其他组织中表达量均很低，几乎不表达。应用I型胶原酶消化法分离获得绵羊内脏和皮下前体脂肪细胞并能被DMEM/F12+5 μg/mL胰岛素+50 μg/mL转铁蛋白质+5 ng/mL亚硒酸钠+50 ng/mL氢化可的松诱导11 d分化为内脏和皮下脂肪细胞；应用II型胶原酶消化法和差速贴壁法分离获得绵羊背最长肌肌内前体脂肪细胞并能被DMEM/F12+0.5 mmol/LIBMX +1 µmol/L DEX+10 mg/L insulin诱导10 d分化为肌内脂肪细胞。3种前体脂肪细胞分化过程中脂肪生成逐渐增加，生长曲线均呈“S”型，第3～9 d为指数生长期。绵羊内脏和皮下前体脂肪细胞中LPL和PPARγ分化第7和11天显著高于第3天，FAS基因第7天最高；而脂联素及其受体基因在两种细胞中的表达水平差异不显著，但三个基因之间AdipoR2表达量最高（35.27），AdipoR1居中（5.24），Adpn最低（0.60）。类似地，绵羊肌内脂肪细胞中AdipoR2表达量极显著高于AdipoR1和Adpn，表达趋势也与3基因在内脏脂肪组织和皮下脂肪组织中相类似，但表达水平有所下降。LPL、PPARγ和FAS在肌内前体脂肪细胞诱导分化过程中的表达模式与内脏和皮下前体脂肪细胞相一致，表明AdipoRs、LPL和PPARγ是脂肪细胞分化的早期标记基因，本项目还初步研究了RNAi沉默脂联素基因对绵羊肌内脂肪细胞生脂的影响。这些材料初步阐明了脂联素介导绵羊内脏、皮下和肌内脂肪细胞生脂的基因表达模式和分子调控机理。