猪肌肉充质干细胞成脂分化的分子机制及其与肌内脂肪生成的关系

Intramuscular fat is a major contributor to meat flavor, meanwhile，it also influences meat color, tenderness and juiciness. In the present application, we proposed that the adipogenic differentiation potential of skeletal muscle stromal stem cells determined the content of intramuscular fat. To prove this hypothesis, we designed series of experiments to illustrate the causality between them using flow cytometry (FCM) and immunohistochemical technique. Subsequently, fragmentation of whole transcriptome of porcine skeletal muscle is to be carried out to characterize the signal pathway underpinning their association. Based on it, pregnant sows, when the offspring skeletal muscle fiber development undergoes a window phase of nutritional regulation, are to be fed with supplementation of nutrients, etc.,conjugated linoleic acids (CLA) and methionine, which are supposed to accelerate the adipogenesis in muscle, to investigate the role of maternal nutritional status in adipogenic differentiation potential of stromal stem cells as well as intramuscular fat accumulation. Thereby, figure out the signal pathway underlying the adipogenic differentiation potential and intramuscular fat deposit, and then identify the key genes/proteins by function analysis approach, to disclose the molecular mechanism of intramuscular fat specifically deposition.

肌内脂肪是肉风味的主要贡献者，同时也影响着肉色、嫩度、多汁性等感官品质。申请者提出肌肉间充质干细胞成脂分化潜力可能决定着猪肌内脂肪沉积水平的科学假设，围绕着解析肌肉间充质干细胞成脂分化-肌内脂肪特异性沉积-肌肉转录组表达谱和miRNA表达谱三者间的逻辑关系这一关键科学问题，设计了利用流式细胞技术和免疫组化技术揭示间充质干细胞成脂分化潜力与猪肌内脂肪沉积的关系，通过Illumina RNA HiSeq技术分析肌肉转录组表达谱和miRNA表达谱，揭示猪肌内脂肪特异性沉积的肌肉生物代谢和信号途径。在此基础上，设计了在母猪妊娠期（后代肌纤维发育的窗口期）日粮中添加调节成脂的共轭亚油酸、作为机体甲基供体的蛋氨酸，研究母体营养对肌肉间充质干细胞成脂分化潜力、肌内脂肪沉积水平的调节作用及其信号途径，通过对信号途径中的关键基因的结构分析、等位基因鉴定和功能验证，揭示肌内脂肪特异性沉积的分子机理。

肌内脂肪是肉风味的主要贡献者，同时也影响着肉色、嫩度、多汁性等感官品质。本项目提出肌肉间充质干细胞成脂分化潜力可能决定着猪肌内脂肪沉积水平的科学假设，围绕着解析肌肉转录组表达谱和基因组甲基化、肌肉组织中成肌/成脂祖细胞分化命运和肌内脂肪沉积潜力三者间的关系这一关键科学问题，利用差速贴壁法分离了肌肉组织中成肌/成脂间充质祖细胞，利用流式细胞技术揭示间成肌/成脂祖细胞的间充质谱系。通过Illumina RNA HiSeq技术和RRBS技术分析了肌肉转录组表达谱和DNA 甲基化谱，研究发现，猪原代成肌或成脂的间充质祖细胞表达谱的结果表明细胞过程和环境信息处理的相互作用在间充质祖细胞成肌或成脂命运决定中发挥重要作用。值得注意的是，我们通过进一步的实验提出了胞外基质与细胞微环境因子的相互作用，通过促进胞内Ca2 + -MLCK和Rho-DMPK信号通路的表达，引起了MLC表达量的上调，这一过程可能通过对细胞骨架的调控最终实现间充质前体细胞向成肌方向的分化而不是成脂方向； RRBS测序结果进一步证明，DNA甲基化通过影响胞外基质与受体的互作介导的细胞骨架的调控来实现间充质干细胞成肌或成脂分化系谱决定的调控作用。本研究在揭示猪肌内脂肪生成细胞分子机制取得了创新性的发现。