CIDEC基因调控奶牛乳腺乳脂合成的机理研究

Milk fat is the major nutrition component in milk and accounts for many of the manufacturing characteristics and organoleptic qualities of milk and milk products. Preliminary study showed that the expression of CIDEC was positively correlated with milk fat content in lactation mammary glands of dairy cows. In cultured mammary epithelial cells, CIDEC knockdown decreased the content of triglycerides, as well as the expression of enzymes and regulatory factors involved in triglyceride synthesis, whereas overexpression of CIDEC had the opposite effects. We also found CIDEC was localized in the nucleus of mammary epithelial cells of dairy cows. All these results suggest that CIDEC has the role in increasing milk fat synthesis by regulating the expression of enzymes related to this process. So in this study, we plan to investigate the effect of acetate and β-hydroxybutyrate on the expression and nuclear accumulation of CIDEC, following by detecting the effect of PI3K-JNK-PPARγ, mTOR and AMPK signal pathways on CIDEC expression. To delineate the regulatory mechanism of CIDEC on the expression of enzymes involved in fat synthesis, coimmunoprecipitation and ChIP were used to detect how CIDEC interacts with C/EBP family members or other transcription factors to regulate the expression of genes encoding enzymes for the milk fat synthesis pathway in mammary gland of dairy cow. These results will help us understand the process of lactation and clarify the molecular mechanism of milk fat synthesis in dairy cows.

乳脂是牛乳的主要成分之一，其含量是关系牛乳营养价值和风味的重要指标。本研究前期结果发现，CIDEC的表达与奶牛的乳脂率之间呈现正相关。奶牛乳腺上皮细胞中CIDEC基因过表达和干扰以后，甘油三酯的合成量发生相应地升高和降低，与甘油三酯合成相关的酶的表达也相应升高和降低。分离纯化奶牛乳腺上皮细胞的核蛋白，检测到CIDEC能够进入细胞核。以上结果表明CIDEC可以通过调节乳脂合成关键酶的表达来调控乳脂合成。为阐明其在乳脂合成调控中的作用方式，我们首先研究乳脂合成前体物对CIDEC表达及转位入核的影响；然后分析PI3K-JNK-PPARγ、mTOR和AMPK等信号通路对CIDEC表达的调控；最后研究CIDEC同C/EBP家族成员或免疫共沉淀技术筛选出的其他转录因子共同作用，调节乳脂合成关键酶表达的调控方式。本研究结果将会使我们更清楚地理解奶牛乳腺的泌乳过程，明确CIDEC调控乳脂合成的分子机制。

乳脂是牛乳的主要成分之一，其含量是关系牛乳营养价值和风味的重要指标。CIDEC是一种脂滴相关蛋白，可以参与脂滴的生物合成过程。本研究前期结果显示CIDEC可以影响乳脂的合成。为进一步阐明CIDEC在泌乳奶牛乳脂合成中的调控方式，本研究首先采用western blot和免疫荧光方法检测乳脂合成前体物是否可以调节CIDEC的表达，然后通过基因干扰和抑制剂处理分析mTOR和AMPK信号通路对CIDEC表达的调控方式，最后研究CIDEC调节乳脂合成关键酶表达的分子机制。结果显示乳脂合成前体物乙酸和β-羟基丁酸可以诱导奶牛乳腺上皮细胞中CIDEC的表达增加，CIDEC可以转位到细胞核，表明CIDEC可以参与泌乳相关基因的调节。信号通路分析结果显示mTORC1和AMPK信号通路可以调节奶牛乳腺上皮细胞中乳脂合成前体物介导的CIDEC的表达。转录因子PPARγ可以通过RXR结合到CIDEC的启动子区，调节CIDEC的转录。脂肪酸合成酶FASN是催化细胞中脂肪酸从头合成的关键酶。荧光素酶报告基因系统和染色质免疫共沉淀结果显示在奶牛乳腺上皮细胞中CIDEC可以与C/EBPβ共同作用，调节FASN的表达，进而提高乳脂的合成。.本研究较好地完成了项目计划书的预定任务，共发表标注本项目资助的SCI研究论文8篇，国家核心期刊论文2篇，超额完成了预期目标。本研究所获结果明确了奶牛乳腺中CIDEC调控乳脂合成的分子机制，可为通过营养调控提高泌乳奶牛乳成分合成提供理论基础和实验依据。