脂肪及其氧化调控对猪克隆胚胎早期发育的影响

Particular high fat content was reported in pig oocytes and the fat was stored in the oocytes as lipid droplets. Even some studies were focused on the survival rate of embryos after cryopreservation by the removal of lipid. However, very limited studies have been carried out on the effect of lipid droplets on the development of pig oocytes and embryos. Therefore, it is very necessary to establish a model to study the effect of lipid droplets on the development of early embryos, and further to research on the functional mechanism. In the present study, combined with centrifugation of lipid droplets and handmade cloning, the cloned embryos with different lipid droplets were constructed to evaluate the effect of lipid on their development. Meanwhile fatty acid oxidation: β-oxidation was evaluated after being modified by L-carnitine and Etomoxir. Finally the in vitro culture system of cloned embryos was improved. The carrying out of this study not only will be benefit to the cryopreservation and in vitro culturing system of pig embryos and oocytes, but also to provide the theoretical knowledge for large-scale production of pigs. It finally provides the animal model for the studies of human diseases such as obesity and fatty liver disease.

猪卵母细胞中含有特别丰富的以球形脂滴形式存在的脂肪。已知这些脂肪的存在影响卵母细胞、胚胎的超低温冷冻保存效果，但是人们对其在猪早期胚胎发育过程中作用还知之甚少。因此，迫切需要通过构建不同脂肪含量的胚胎，进而深入研究脂肪对猪早期胚胎发育的影响极其分子机制。本课题拟借助离心去脂和手工克隆技术，建立不同含量的体细胞克隆胚胎，追踪脂肪在胚胎中的动态变化，检测不同脂肪含量对胚胎能量代谢及发育命运的影响。同时，通过L左旋肉碱和Etomoxir调控克隆胚胎中脂肪的β-氧化，进一步改善和优化不同脂肪含量胚胎的体外培养体系，促进胚胎发育，以揭示脂肪对猪早期胚胎发育的可能的作用机制。本研究将有助于增进人们对脂肪参与胚胎发育的了解，也可为完善猪胚胎超低温保存、体外培养体系提供依据，对加速猪胚胎工程更好地服务于畜牧良种扩繁、人类疾病动物模型制备以及辅助生殖具有重要意义。

猪卵母细胞中含有特别丰富的脂滴。一般认为脂滴是细胞中一种能量存储形式，为早期胚胎发育提供主要的能量供应。而近几年的研究主要集中在脂滴对胚胎冷冻效率的研究，但对猪卵和胚胎中脂滴代谢及其作用机制研究并不多。本文首先利用两种方法构建不同脂滴含量的猪胚胎，第一种则是结合手工克隆中的切割、半卵融合技术与卵母细胞离心去脂的方法构建不同脂含量的猪胚胎，第二种则是利用RNA干扰的方法敲减调控甘油三酯合成的关键基因LIPIN1，从而降低胚胎中的脂肪。随后分析这些胚胎中脂滴的动态分布、与脂滴代谢有直接关系的线粒体与内质网等的分布，结果证明，去脂胚胎中的细胞器都受到非常显著的影响；其次分析去脂对胚胎中脂肪酸氧化的变化、能量ATP代谢、关键基因表达变化、关键蛋白或酶的动态变化等，结果证明，低脂不仅仅影响了猪早期胚胎发育，而且对其发育过程中一些关键基因及蛋白都有显著影响。更进一步利用L-左旋肉碱与Etomoxir来调控猪胚胎中脂肪的β氧化，进而改善低脂胚胎的发育效率及其冷冻效果，建立具有较高效率的猪胚胎冷冻体系。最后，生产不同脂含量的胚胎，进行胚胎移植，获得克隆小猪，为更深入研究提供基础，此外结合本课题开展，自主设计并生产了一些胚胎实验专用的小设备并申请专利，其中主要包括卵的手工切割刀、胚胎移植箱、RNA干扰设备及新技术等。