线粒体网络重建在猪前脂肪细胞分化及脂肪沉积中的作用研究

Regulation of porcine preadipocyte differentiation can effectively improve carcass lean and the pork quality. Mitochondrial network (including number, morphology, and distribution) is essential for the maintenance of adipocyte function. And the transformation of mitochondrial network status (mitochondrial network reconstruction) is a key factor in determining the efficiency of adipocyte differentiation. Research reveales that the number of mitochondria in porcine adipocyte differentiation and maturation is dynamically changed. However, the dynamic process of porcine adipocyte mitochondria reconstruction (including fusion, fission, and autophagy) is still unclear. Therefore, we aim to analyze the maturation process of porcine adipocyte from a new perspective of mitochondrial network. To this end, we plan to use molecular detection and immune electron microscopy to determine the dynamic changes during pig preadipocyte differentiation, including the number, morphology, distribution, oxygen consumption of mitochondria and mitochondrial membrane potential. Analysis of the expression of fission-, fusion-, and autophagy-related proteins in the progress of pig preadipocyte differentiation and the adipose tissue of different pig breeds, to identify the expression "peaks" of related-proteins. The associated activator or inhibitor of the mitochondrial fusion, fission and mitophagy is added in medium to discover the effect on the process of pig preadipocyte maturation. This study would provide a new theoretical basis for the mechanisms of porcine adipocyte differentiation and fat deposition.

调控猪脂肪细胞分化能够有效提高胴体瘦肉率和改善猪肉品质。线粒体网络（数量、形态、分布）对维持脂肪细胞功能至关重要，且线粒体网络状态的转变（线粒体网络重建）是决定脂肪细胞分化效率的关键因素。研究发现线粒体数目在猪脂肪细胞分化成熟的过程中呈动态变化，但猪脂肪细胞线粒体网络重建的动力学过程（融合、分裂与自噬）尚不清楚，因此申请人拟从线粒体网络的全新视角解析猪脂肪细胞的成熟过程。本项目拟通过分子检测和免疫电镜观察，阐明线粒体在猪前脂肪细胞分化过程中的动态变化过程，包括线粒体的数量、形态、分布、耗氧量和线粒体膜电位；分析猪前脂肪细胞分化及中外猪种成熟脂肪组织线粒体分裂融合和线粒体自噬蛋白的表达，确定分化过程线粒体分裂融合和自噬发生的“高峰期”；给予线粒体融合分裂和线粒体自噬相关的激活和抑制分子，研究其对猪前脂肪细胞分化成熟的影响，为探讨新的猪脂肪细胞分化和脂肪沉积机制提供理论基础。

脂肪发育调控是改善猪胴体瘦肉率和肉质的重要研究方向，线粒体为脂肪分化发育提供了能量和物质基础，然而不同类型脂肪细胞的线粒体的分布、形态及代谢等特征还未完全阐明。第一，本项目阐明猪不同类型脂肪细胞（白色和米色）的线粒体形态、分布和膜电位等特征，发现它们的膜电位没有明显不同，白色脂肪细胞线粒体主要分布在脂滴周围，而米色脂肪细胞线粒体未包围脂滴；第二，本项目揭示了不同类型脂肪细胞重塑过程中线粒体融合、分裂、自噬及其代谢变化，发现白色脂肪米色化主要增强线粒体β氧化，而褐色脂肪白色化使线粒体融合、分裂、自噬均减少，并伴随着脊减少；第三，发现外源解耦联蛋白UCP1增加了猪脂肪组织线粒脊的形成，并且同家族基因UCP3的核心启动子在藏猪脂肪组织甲基化比例高于巴马猪；第四、鉴定了泛素连接酶RNF20介导的H2B单泛素化对白色和褐色脂肪分化发育、线粒体网络重建及脂代谢的影响，RNF20缺失通过影响H3K4me3和H3K79me3的修饰抑制白色和褐色脂肪细胞分化，并且RNF20缺失是体内褐色脂肪组织发生白色化，伴随着线粒体出现肿胀、嵴消失和空泡化等特征；最后，对白色化的褐色脂肪进行脂质组分析，发现中性脂类显著的升高，而磷脂类显著的下降，心磷脂C176:13(18:2)下降最显著，提示其可能线粒体网络重建的重要代谢分子。本项目的研究结果表明线粒体网络重建是脂肪发育和重塑过程的关键特征，鉴定到RNF20是调控猪脂肪沉积的新靶点，心磷脂C176:13(18:2)可作为指示线粒体功能的重要脂质代谢分子。