猪Cidec调控脂滴融合及其表达调控的机制研究

Fat deposition has been a key issue in the research of pig genetics and breeding. Lipid droplets is the main storage form of lipid in cells, and the mechanism of its development is still unclear. In recent years, it has been found that the Cidec of human and mouse has a direct effect on triglycerides accumulation and lipid droplets fusion in adipose tissue and liver. Cidec can promote lipid droplet fusion interacted with Cidea, Plin1 and other factors, and its promoter is regulated by transcription factors such as PPAR, CEBP and so on. We and others have shown that the Cidec of Porcine can promote lipid droplet fusion and interact with ADRP. In addition, we found that Cidec is increased under the high fat and fasting conditions. Accordingly, we propose that Cidec is regulated by post transcriptional regulation and Cidec can interact with ADRP and other proteins to promote lipid droplet fusion. Therefore, using high-throughput sequencing and fast cultivation of yeast two hybrid method, this study makes clearly which miRNA to regulate cidec and how to regulate under the condition of fast culture, and it will be clear whether Cidec promotes lipid droplet fusion by interacting with ADRP and other proteins.. This study will provide a new theory for lipid metabolism in Porcines.

脂肪沉积一直是猪遗传育种研究的关键问题。脂滴是脂肪沉积的主要形式，其发育调控机制仍不清晰。近些年发现，人和小鼠的Cidec在脂肪组织和肝脏中对甘油三脂积累和脂滴融合有直接作用，并且发现Cidec可与Cidea、Plin1等因子相互作用促进脂滴融合，其启动子受PPARα、CEBP等转录因子调控。但本课题组初步证实猪Cidec与ADRP相互作用，且促进脂滴的融合和甘油三酯的积累；另外发现高脂和禁食培养条件下猪Cidec表达量均增加。据此提出假设：不同营养条件下，Cidec受到不同转录因子调控；且Cidec可与ADRP等蛋白互作促进脂滴融合。为此，本研究利用染色质免疫共沉淀及酵母双杂交等方法明确不同营养条件下，Cidec受哪些转录因子调控及具体机制，且本研究将明确Cidec促进脂滴融合是否通过与ADRP等蛋白互作引起。本研究的开展将为猪脂类代谢的提供新理论。

本项目采用不同禁食时间处理普通和肥胖小鼠，比较肝脏组织解剖学、生理生化指标等方面变化，然后分析肝脏脂滴形态及脂质代谢相关基因表达活性差异；在此基础上采用RNA-seq技术研究禁食对肝脏转录水平的影响，进一步选择对脂滴融合起关键作用的Cidec作为研究对象，从Cidec上游转录因子、信号通路以及miRNA等角度，分析禁食对肝脏Cidec表达调控的分子机制。.结果显示：随着禁食时间延长，小鼠体重、肝脏指数均下降，且在肥胖小鼠中该现象更加明显；肥胖小鼠在禁食后血液指标较普通小鼠波动大，葡萄糖含量下降，游离脂肪酸含量、甘油三酯含量总体呈上升趋势，LDL和胆固醇含量呈现下降趋势。利用组织切片技术，通过组织油红O染色方法观察肝脏组织中脂滴的形态变化。结果显示禁食后小鼠肝脏切片出现脂滴体积增大现象。利用RNA-Seq技术分析禁食后转录组变化情况，结果显示这些差异表达基因显著富集到免疫反应、细胞骨架和脂肪代谢等相关生物学过程中。通过特异性分析脂质沉积相关基因表达量变化，发现Cidec在肥胖小鼠中表达量极高。禁食后Cidec的mRNA水平和蛋白质水平均发生显著升高，Cidec过表达的细胞中脂滴体积明显增大，脂滴个数减少。随着禁食时间的延长，AMPK及其磷酸化水平不断上升。进一步分析AMPK下游促进脂滴融合相关的转录因子，发现PPARγ表达量增加，利用双荧光素报告系统发现PPARγ提高了Cidec启动子荧光活性。利用生物信息学方法预测靶向调控Cidec的候选miRNA，结果发现miR-7a-5p可以靶向结合Cidec的3’-UTR，利用Cidec的3’-UTR突变技术表明miR-7a-5p抑制Cidec的表达。本项目为禁食情况下肝脏脂肪沉积的研究提供了一个新的途径。.