基于Cre/loxP系统的肝特异性表达REGγ转基因小鼠的建立及脂质代谢分析

Hepatic steatosis resulting from the excessive lipid accumulation in liver has been one of the high incidence diseases in today’s society, however the pathogenesis underlying it has yet not fully elucidated. Our previous studies showed that systemic REGγ (the proteasome activators) deficiency can protect against high-fat-diet induced fatty liver in mice. However, it is still unknown whether or not such regulation is hepatic cell autonomous effect. We will construct pCMV-loxp-stop-loxp-flag-REGγ expression vector and the constructs will be introduced into the fertilized eggs by microinjection to obtain the transgenic mice. Then the transgenic mice will be crossed with the Alb-cre transgenic mice to obtain the LSL-Flag-REGγ;Alb-Cre double transgenic mice.The double transgenic mice will then be crossed with REGγ-/- mice to generate the hepatocyte specific REGγ transgenic mice in overall REGγ deficient background. We will analyze the lipid metabolism in mice liver after 19 weeks HFD feeding to explore if REGγ regulates lipid metabolism in hepatic cell autonomous mechanism. We will further investigate the mechanism whether the authophagy play a role in lipid metabolism of liver tissue.

脂质过度积累导致的脂肪肝是当今严重威胁人类健康的高发疾病之一，然而其发病机制尚未完全阐明。我们的前期研究表明全身性的蛋白酶体激活因子REGγ基因缺失通过自嗜抑制高脂食物引起小鼠脂肪肝，但是该调控是否为肝脏细胞自主性方式还不得而知？ 本课题通过构建pCMV-loxp-stop-loxp-flag-REGγ表达载体，采用受精卵显微注射的方式获得转基因小鼠，然后与肝脏特异性表达Cre的Alb-Cre转基因小鼠杂交，获得LSL-Flag-REGγ; Alb-Cre 双转基因小鼠，该小鼠通过与REGγ-/-小鼠杂交，得到REGγ敲除背景的肝脏特异性表达REGγ的转基因小鼠。高脂食物喂养19周后对各组小鼠肝脏脂质代谢分析，探讨REGγ对肝脏脂质代谢是否为肝脏细胞自主性（hepatic cell autonomous）调控，同时从分子机制上探讨自嗜是否参与了REGγ对肝脏成脂的细胞自主性调控。

课题建立了基于Cre/loxP 系统的肝特异性非表达REGγ 转基因小鼠，为研究REGγ调控机体的代谢水平奠定基础。表型研究发现：REGγ基因缺失促进外周组织肌肉中脂质代谢，包括脂肪酸氧化、电子传递已经氧化磷酸化的进行，进而提高小鼠的运动水平及耐力。机制的研究表明：REGγ通过负调控LKB1蛋白水平，调控下游的能量感应蛋白AMPK的蛋白活性，进而调控脂质代谢关键的转录因子PGC1α的活性以及下游的基因表达，从而实现对脂质代谢及能量代谢的调控。研究结果表明REGγ有可能作为治疗机体脂质代谢乃至代谢综合征疾病的靶点。