Txnip在猪前体脂肪细胞分化中的作用及机理研究

Thioredoxin interacting protein (Txnip) is a multifunctional protein involved in many cellular and physiological processes, and can regulate cellular redox state by inhibiting the activity of thioredoxin. The former researches suggested that Txnip degradation or Txnip expression down-regulation is required for adipocyte differentiation. Glucose stimulates Txnip expression in adipocytes through transcriptal factor carbohydrate-response element-binding protein (ChREBP) which binding to the carbohydrate-response elements (ChoREs) on the Txnip promoter. 1alpha, 25-(OH)(2)-VD3 up-regulated Txnip expression and inhibit adipogenesis. Carbohydrate, which is the main substrate of lipogenesis and energy source for pigs, promote adipocyte differentiation, adipogenesis and lipogenic gene expression through ChREBP mediated by its intermediated product glucose-6-phosphate as molecular signal. Therefore, to explore effects of Txnip on porcine preadipocyte differentiation and its mechanism, porcine Txnip lentivirus interfering vector Lenti-Txnip-shRNA and Txnip recombinant adenovirus vector pAdTrack-Txnip are to be constructed. Lenti-Txnip-shRNA transfected preadipocytes and non transfected preadipocytes will be treated with different concentration glucose and 1alpha, 25-(OH)(2)-VD3 in the adipogenic medium separately. The pAdTrack-Txnip transfected preadipocytes and non transfected preadipocytes cultured in the adipogenic medium, are to be treated with PD98059 or U0126, the ERK-specific inhibitor, to inhibit the activation of extracellular signal-regulated kinase (ERK) signaling pathway. At the different phase of adipogenesis, the morphological characters will be detected by Oil red O staining. Triglyceride contents will be detected by Triglyceride E-test Wako kit. The activations of ERK1/2 molecular signaling pathway (MEK1/2、ERK1/2、p-ERK1/2) and the expressions of lipogenic gene (C/EBPα、C/EBPβ、PPARγ、ap2、Perilipin) are to be analyzed by real time qRT-PCR and western blot methods. These results will elucidate the role and molecular mechanism underlying regulation of Txnip in porcine preadipocytes differentiation and provide theoretical foundation for understanding of adipogenesis mechanism and control of lipid accretion and improving meat quality.

硫氧还蛋白互作蛋白（Txnip）是一种氧化还原调节蛋白，研究推测Txnip降解或表达下调可能是脂肪细胞分化的必要条件。葡萄糖通过转录因子ChREBP与Txnip基因启动子上ChoRE结合，促进脂肪细胞Txnip表达；1, 25-(OH)(2)-VD3上调Txnip表达，抑制脂肪细胞分化。碳水化合物是单胃杂食动物主要能量来源和生脂底物，以其代谢中间产物葡萄糖-6-磷酸为信号分子，通过ChREBP促进脂肪细胞分化，调控生脂及生脂基因表达。因此，本项目拟以分离培养的八眉猪前体脂肪细胞为材料，构建和转染靶向Txnip的siRNA干扰和超表达载体，分别用葡萄糖和VD3处理Txnip表达沉默细胞，ERK抑制剂处理Txnip过表达细胞，通过形态观察、分析脂肪生成、ERK信号通路及生脂关联基因表达的变化，研究Txnip在脂肪细胞分化中的作用及调控机制，为揭示猪脂肪细胞分化的分子调节机制奠定基础。

脂肪组织主要由脂肪细胞组成。脂肪组织发育过程中脂肪细胞数量过度增加和（或）体积过度增大，导致人肥胖发生或动物体脂过度沉积而影响生产效益。因此，阐释脂肪细胞分化的调控机制有十分重要的意义。本项目利用原代培养猪脂肪细胞，研究了不同经济类型猪种八眉猪和长白猪皮下和肌内脂肪细胞增殖分化的差异及其对主要成脂调控因子葡萄糖和胰岛素的应答；利用分子生物学技术，通过沉默和过表达氧化还原调节蛋白Txnip，研究证明了Txnip可通过下调成脂分化关键因子PPARγ表达，抑制猪前体脂肪细胞分化；葡萄糖促进猪前体脂肪细胞分化，Txnip可抑制葡萄糖对细胞分化的促进作用；1,25(OH)2VD3对猪前体脂肪细胞增殖和分化具有双向作用，低浓度1,25(OH)2VD3可通过上调Txnip基因表达抑制猪前体脂肪细胞分化；探讨了猪前体脂肪细胞分化过程中Txnip与ERK1/2 信号通路关系，Txnip表达沉默提高了ERK1/2表达，但Txnip过表达对没有影响ERK1/2表达，ERK1/2活性变化对Txnip表达没有影响。全面完成了项目要达到的研究目标。项目研究结果对于认识葡萄糖、VD调控猪脂肪形成的机制及通过营养控制脂肪沉积具有重要的学术价值，为最终阐明猪脂肪沉积的分子机制提供了理论依据。