猪肌肉外泌体（Exosome）介导脂肪生成及信号传递的分子机制

Exosome as a new cellular messenge has become research hotspot. It is secreted by the cells and involved in the transmission of information and the functional regulation of tissue or cells via autocrine, paracrine, and endocrine pathways. Currently the research of exosomes in animal is little known, especially in the pig. Our early studies have shown that pig muscle exosomes may be involved in the development of adipose tissue, mediation of the cross-talk between muscle and fat tissues, but the mechanism is unclear. This study, using primary cell model and molecular technique of over-expression, RNA interference, target knockout, blotting, immune-precipitation, tracer technique and so on, will investigate the function mechanism of muscle exosomes on proliferation and differentiation in adipose precursor cells, and reveal the candidate molecule in exosomes and key signal target in mediating lipogenesis, especially the targeting modification of the exosome miRNA. On above basis, further explore the formation mechanism of exosomes using key regulator such as cytokine and fatty acid to treat adipose precursor cells, and observe its function in vivo for regulating intramuscular adipogenesis by intramuscular injection. This study will reveal that the molecular mechanism of cross-talk between porcine muscle and fat through mRNA-miRNA-protein regulatory networks mediated by exosomes. After that, there will be a new perspective for the mechanism of the porcine meat quality formation and important targets for health regulation in swine with diet-induced metabolic syndrome.

外泌体作为一种新型“细胞信使”已成研究热点。由细胞分泌，通过自分泌、旁分泌和内分泌方式参与组织或细胞间的信息传递与功能调节。目前在动物上研究不多，尤其在猪上。课题组前期研究显示，猪肌肉组织可能通过“外泌体信号”参与了脂肪生成的调节，但机制还不清楚。本项目拟运用原代细胞模型，采用过表达、干扰、敲除、印迹、示踪以及免疫共沉淀等分子手段，揭示肌肉外泌体对脂肪前体细胞增殖与分化的机制，解析调节脂肪生成的候选分子和关键信号靶点，尤其是肌肉外泌体中miRNA的修饰调节作用。在此基础上，进一步探讨重要调节物对肌肉外泌体形成及其功能的调控，以及外泌体在活体环境下的示踪及调节脂肪生成的作用。本研究将从细胞、调节物和活体水平上系统阐明肌肉外泌体所介导的mRNA-miRNA-蛋白质对脂肪生成的分子机制，以新的角度为肌肉与脂肪互作理论提供突破性资料，为猪肉品质形成以及猪营养代谢症的健康调控提供新的重要靶标和素材。

肌肉和脂肪组织间互作是影响猪胴体产量和质量的重要基础，其中肌肉-脂肪信号轴是调节肌肉和脂肪组织间互作的主要机制。外泌体作为一种天然活性物质运输载体和新型疾病诊断标记物，已成为新的研究热点。由细胞分泌，参与组织或细胞间的信息传递与功能调节。课题组前期研究显示，猪肌肉组织可能通过“外泌体信号”参与脂肪生成，介导肌肉与脂肪间的对话，但其调节机制还不清楚，尤其是外泌体中miRNA的调节作用。本项目首先对猪源肌肉外泌体与脂肪外泌体进行miRNA高通量测序，筛选肌肉外泌体中有差异表达的关键miRNA；接着运用组织特异性miRNA基因缺失动物以及原代细胞模型，采用过表达、干扰、敲除、印迹、示踪以及免疫共沉淀等分子手段，研究揭示了猪肌肉组织可以通过其分泌的外泌体途径抑制脂肪细胞的增殖与分化的机制；解析了胰岛素或油酸可以通过肌肉细胞外泌体的新途径来调节脂肪细胞的分化聚酯；发现了猪肌肉外泌体中的miR-146a可能是抑制脂肪细胞分化聚酯的关键miRNA，阐明了miR-146a-5p 通过靶向 GDF5 调控 PPARγ 信号通路调节脂肪生成的分子机制，且肌肉 miR-146a-5p 特异性敲除可以促进动物模型体内的脂肪沉积。本项目研究从体外和体内水平，层层递进地阐明了肌肉外泌体 miR-146a-5p 作为新脂肪-肌肉因子参与肌肉-脂肪信号轴，进而调节脂肪生成的分子机制，为研究肌肉和脂肪互作关系提供了新的代谢机制与靶标，同时也为猪肉品质形成以及猪营养代谢症的健康调控提供了新的理论依据。