SORT1介导载脂蛋白B依赖自噬降解调控鸡肝细胞脂代谢分子机制研究

Abdominal fat deposition in poultry will reduces meat quality and growth rate and one of the inducers of abdominal fat deposition are disorders of liver lipid metabolism. Although Apolipoprotein B synthesizes VLDL through its self-degradation and participates in the regulation of hepatic lipid metabolism, it is unclear that whether it depends on autophagy in lipid metabolism and whether it requires another factors to regulate this process. Recently, we found starvation induces autophagy initiation and up-regulate apoB expression which means there was a interaction between autophagy and apoB. However the specific regulatory mechanism was not clear. In this study we will analysis ApoB-dependent mechanisms of autophagy degradation by 3-MA to treated chicken hepatocyte. And discuss the interaction between SORT1 and ApoB through CO-IP. Further we will detect the mechanism of SORT1 mediates the ApoB-dependent autophagy degradation via immunofluorescence, flow cytometry and western blot. This project will reveal the effect of ApoB on lipid metabolism in chicken hepatocytes, improve liver lipid metabolism in poultry, and provide molecular basis for improving poultry feed efficiency and reducing production costs in poultry breeding.

禽类腹脂过度沉积会降低肉品质及生长率，肝脏脂代谢失调是引起腹脂沉积的诱因之一。载脂蛋白B通过自身降解合成VLDL参与调控肝脏脂质代谢，但其是否依赖自噬参与脂代谢以及是否需要第三方因子调控该过程的分子机制尚不清楚。本课题前期结果发现鸡饥饿诱发自噬启动并上调apoB的表达，表明二者有潜在联系，但具体机制尚不清楚。本项目将利用3-MA处理鸡肝细胞，分析ApoB依赖自噬降解的机制；采用CO-IP检测SORT1与ApoB的关系，解析SORT1与ApoB的互作方式；利用细胞免疫荧光、流式细胞分析与蛋白免疫印迹进一步探讨SORT1介导ApoB依赖自噬降解的作用机制，深入探究ApoB、SORT1与自噬三者对鸡肝细胞脂代谢调控影响脂质沉积的机制。研究结果将全面揭示ApoB对鸡肝细胞脂代谢影响，完善禽类肝脏脂代谢过程，为提高禽类饲料利用率、降低养殖业生产成本提供理论依据。

禽类腹脂过度沉积会降低肉品质及生长率，肝脏脂代谢失调是引起腹脂沉积的诱因之一。载脂蛋白B通过自身降解合成VLDL参与调控肝脏脂质代谢，但其是否依赖自噬参与脂代谢以及是否需要第三方因子调控该过程的分子机制尚不清楚。本项目通过构建ApoB基因、Atg5基因、Sort1基因的过表达载体、干扰与敲除载体，综合分析了ApoB、Sort1与自噬三者相互调控的关系，同时摸索出3-甲基腺嘌呤处理鸡肝细胞的最佳条件，进一步揭示自噬与ApoB的关系。并利用饥饿实验揭示个体水平中鸡肝脏自噬与脂代谢的关系，结合二代测序技术深入挖掘鸡肝脏内饥饿诱发自噬活动启动后，受自噬调控的关键脂代谢基因与信号通路。结果显示，鸡肝脏内存在自噬活动，且饥饿可诱发鸡肝脏自噬活动的启动，饥饿导致鸡血清内葡萄糖与甘油三酯水平下降，补饲后回升；同时，饥饿可促使鸡肝脏内脂代谢基因与自噬相关基因mRNA水平与蛋白水平发生变化；随饥饿时间的增加，鸡肝脏内自噬小体数目增加。另外，自噬过程受到抑制后，导致脂代谢基因mRNA表达量上调，过表达Atg5基因后ApoB的mRNA水平降低；Sort-1的mRNA水平显著增加，FABP7无变化；干扰Atg5后，ApoB、FABP7的相对表达量与对照组相比显著升高，Sort-1无变化，由此揭示Atg5参与脂质代谢，并且与ApoB、FABP7、Sort1存在调控关系。通过转录组测序技术对24小时和72小时样品进行测序分析获得以下关键基因：SREBF2、TERAL2、VCP、SREBF1和LOC100858796；关键信号通路如下：p53 signalling pathway、Toll-like Receptor signalling pathway、Autophagy、Lysosome、Mitophagy、MTOR signalling、脂肪酸降解、脂肪酸生物合成和不饱和脂肪酸生物合成。此结果揭示自噬与ApoB对鸡肝细胞脂代谢影响，完善禽类肝脏脂代谢过程，为提高禽类饲料利用率、降低养殖业生产成本提供理论依据。.

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