维生素D对猪前体脂肪细胞增殖分化的影响及机制研究

Adipogenesis involves the proliferation and differentiation of preadipocytes, which are affected by Vitamin D as nutrient and hormone factor. However, there are discrepancies in these existing results that might be affected by species, genotypes and cell types. Bamei and Landrace pigs are differed visibly in fat deposition. Therefore, in this study, Bamei and Landrace pigs in different growth stages will be used in order to study the relationship between Vitamin D and adipogenesis by detecting the content of Vitamin D and its metabolic enzyme activity and measuring the number change of preadipoctyes, as well as the number and size change of adipocytes in subcutaneous adipose tissue, visceral adipose tissue and longissimus dorsi. Intramuscular and subcutaneous preadipocytes of Bamei and Landrace pigs will be isolated and cultured. The role of Vitamin D on proliferation and differentiation of preadipocytes and the discrepancies between intramuscular and subcutaneous adipocytes of these different pig breeds will be studied. The expression of Insig2a or Txnip genes is being silenced or over-expressed in the preadipocytes, and then the cells are treated with different concentration of 1 alpha,25-dihydroxyvitamin D3. The metabolic enzyme activity of Vitamin D, the gene expression of key transcriptional factor related to adipogenesis and the cellular redox will be analyzed to explore the role of Insig2a (Insulin-induced genes, Insigs) and Txnip/Trx (thioredoxin interacting protein/thioredoxin) redox system in the differentiation of porcine preadipocytes, thereby explore the mechanism of Vitamin D on porcine preadipocytes differentiation. This study is important for providing theoretical basis that clarify the mechanism of adipogenesis.

脂肪细胞增殖和分化是脂肪形成的基础。VitD作为营养及激素类物质，影响脂肪细胞增殖分化。然而，已有的研究结果有明显争议，且存在物种、基因型及组织细胞差异。因此，本项目以脂肪沉积能力明显不同的八眉猪和长白猪为研究对象，检测其不同生长阶段肌肉和皮下、内脏脂肪组织VitD含量及其代谢酶表达、前体脂肪细胞数目及脂肪细胞数目和大小的变化，比较研究VitD代谢的组织、时序变化，阐明体内VitD与脂肪形成的关系；采用细胞培养技术，体外研究VitD对猪皮下和肌内前体脂肪细胞增殖分化的影响及其在猪种间差异，并应用RNAi和超表达技术，沉默或过表达Insig2a和Txnip基因，分析其对VitD代谢酶、脂肪细胞分化关键蛋白基因表达、细胞氧化还原平衡的影响，研究Insig2a和Txnip/Trx氧化还原系统在猪前体脂肪细胞分化中的作用，探讨VitD影响猪前体脂肪细胞分化的机理，为阐明脂肪形成的机制提供理论依据。

脂肪组织主要由脂肪细胞组成，脂肪细胞增殖和分化是脂肪形成的基础。猪生长过程中皮下脂肪和肌内脂肪含量的变化主要是脂肪细胞肥大的结果，八眉猪生长发育过程中脂肪细胞体积比长白猪有更大的增加。脂肪组织是VitD的贮存器官，且具有代谢VitD的作用。作为营养及激素类物质，VitD对猪前体脂肪细胞增殖分化具有双向作用，低浓度（0.1 nmol/L）促进猪前体脂肪细胞增殖、抑制分化，而高浓度（100 nmol/L）抑制猪前体脂肪细胞增殖、促进分化。VitD对猪前体脂肪细胞分化的影响是个持续的过程，且在猪前体脂肪细胞分化中期作用最为显著。Insig2a表达沉默促进猪前体脂肪细胞分化，而Insig2a过表达抑制猪前体脂肪细胞分化，但Insig2a可能不是VitD调控脂肪细胞分化的主要介导者。VitD可能是通过调节Txnip/Trx表达，影响ROS产生，改变细胞氧化还原状态，从而调控猪前体脂肪细胞分化。VitD也可通过调节SRSF1的表达进而影响PPARγ的选择性剪接，PPARγ∆5可能通过下调PPARγ的表达量来调控Txnip及成脂相关基因，进而抑制脂肪细胞的分化。研究结果对于认识VitD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，奠定了生产应用基础。