线粒体解偶联蛋白（UCP）在猪卵母细胞脂质代谢与冷冻中关键作用研究

卵母细胞体外成熟和胚胎体外发育过程中，培养基内血清类物质会造成脂肪的异常沉积，导致卵母细胞成熟和胚胎发育能力降低，尤其是抗冻性变差。现已发现卵母细胞脂肪代谢异常会直接影响线粒体功能。我们前期研究已发现体内外成熟卵母细胞的差异表达基因主要集中在线粒体内膜蛋白上，但脂肪代谢影响线粒体功能的机制尤其是内膜解偶联蛋白UCP关键调控作用亟待深入研究。本研究拟通过调控体外成熟过程中猪卵母细胞脂质代谢，结合解析电喷离子化质谱分析技术(DESI-MS)、免疫荧光染色和激光共聚焦显微镜等手段，分析脂质含量、组分变化；采用qRT-PCR，WB，酶标仪及透射电镜等方法检测线粒体的拷贝数、膜电势、代谢温度、超微结构和UCP基因蛋白表达等变化，并通过TMT蛋白质全谱（Shotgun）技术获得不同脂质代谢水平下线粒体靶向蛋白表达谱进行分析。研究力图为进一步改善体外培养体系，提高细胞发育能力和冷冻耐受性提供理论基础。

Excess lipids are accumulated when oocytes and embryos that undergo in vitro maturation/ production (IVM/ IVP), which lead to the comprised efficiency of maturation, development and cryopreservation. Previous study proved that abnormal oocyte lipid metabolism affected mitochondrial function during in vitro maturation. We found that the differentially expressed genes of in vitro mature oocytes were concentrated in mitochondrial inner membrane proteins. At present, the mechanism study of lipid metabolism affecting mitochondrial function especially the key role of uncoupling protein (UCP) is needed. In the present study, lipid metabolism were modulated during in vitro maturation, and adopts electrospray ionization mass spectrometry analysis techniques (DESI-MS), immunofluorescence staining combined with confocal laser scanning microscopy were used to analysis the content and composition of cytoplasmic lipid droplets. Real Time-qPCR,WB, multiscan spectrum and transmission electron microscope and so on were used to detect the mitochondria DNA copy numbers, mitochondria membrane potential, mitochondrial temperature, ultrastructure and the expression of UCP. TMT-Shotgun-MS were used to obtain and analysis proteome of the mitochondrial targeted protein to illustrate the mitochondrial function under different lipid metabolism condition. This study is trying to disclose the influence of lipid metabolism on mitochondrial function especially the key role of UCP and provide theoretical basis for optimizing in vitro culture system to improve oocyte and embryo viability and cryotolerence.