肠特异性CGI-58基因敲除致小鼠脂质代谢紊乱的研究

CGI-58, a member of α/β-hydrolase fold family, appears to function as a coactivator of adipose triglyceride lipase (ATGL), playing a vital role in cellular lipid metabolic homeostasis. Whole body CGI-58 knockout （KO） mice die prematurely due to skin barrier dysfunction, preventing the further research about its physiological functions. It is necessary to figure out its lipid metabolic mechanism in the body since mutations in CGI-58 cause Chanarin-Dorfman Syndrome (CDS), an autosomal recessive disease characterized by the storage of triglyceride -rich lipid droplets (LDs) in most cells. At present, we successedly create the CGI-58 condition KO mice (floxed mice) and generate the intestinal-specific KO mice after crossing these floxed mice with Villin-Cre mice. The pilot data show that intestinal-specific CGI-58 KO directly causes lipid accumulation in the intestine, illustrating its important role in intestinal lipid metabolism. Thus, on the basis of preliminary studies, the overall goal of this proposed study is to define the physiological role of CGI-58 in intestinal lipid metabolism and explore the detailed mechanisms in this process. Also, we will investigate whether CGI-58 KO impairs the CM secretion and cholesterol absorption, eventually providing a theoretical basis about the prevention and treatment of this disease.

Comparative Gene Identification-58 （CGI-58）, 也被称为α/β-水解酶结构域包含蛋白5, 是甘油三脂水解酶的激活因子，在细胞脂代谢平衡中起重要作用。CGI-58基因敲除小鼠由于皮肤屏障缺陷，出生后不久即死亡，制约了对其生理功能的研究。鉴于CGI-58突变 可导致脂质贮积症，因此有必要进一步明确CGI-58在体内脂代谢的作用。目前我们已经成功建立了CGI-58 条件性基因敲除 (floxed)小鼠，与Villin-Cre小鼠交配后，得到肠特异性CGI-58敲除小鼠。初步结果表明大量脂滴沉积在小鼠肠粘膜上皮细胞内，提示CGI-58在小肠脂质代谢中具有重要的作用。本项目将在前期研究的基础上，阐明CGI-58在肠粘膜上皮细胞中的生理功能，明确其具体分子机制，并探讨CGI-58敲除后对乳糜微粒分泌和胆固醇吸收的影响，这将为该类疾病的防治提供理论依据。

CGI-58是脂肪甘油三酯水解酶（ATGL）的激活因子，能有效的促进ATGL介导的甘油三酯分解代谢。先前的研究表明，CGI-58在消化道胆固醇代谢中发挥生物学功能，而且该功能独立于其对ATGL的激活作用。由此，我们大胆提出假说，CGI-58参与了巨噬细胞胆固醇代谢及炎症反应，并因此影响动脉粥样硬化的发生发展。本课题中，我们应用SRA启动子建立了巨噬细胞特异性过表达的CGI-58小鼠（Mac-CGI-58 Tg），该小鼠进一步与ApoE−/−杂交，建立巨噬细胞特异性CGI-58过表达的ApoE缺失小鼠（CGI-58 Tg/ApoE−/−）。各品系小鼠，包括Mac-CGI-58 Tg小鼠及其对照的巨噬细胞CGI-58正常表达的ApoE缺失小鼠（CGI-58 WT/ApoE−/−），Mac-CGI-58 Tg 小鼠和Mac-CGI-58 WT小鼠，用高脂饲料喂养12-18周。同时培养RAW264.7巨噬细胞系，用细菌脂多糖（lipopolysaccharide；LPS）处理。检测巨噬细胞胆固醇及脂质代谢水平；检测巨噬细胞炎症反应及其活性氧的生成和线粒体功能；检测过氧化物酶体增殖受体（PPAR）通路；检测主动脉粥样硬化各表型指标。实验结果表明相对于CGI-58 WT/ApoE−/−对照小鼠，CGI-58 Tg/ApoE−/−动脉粥样硬化病变程度显著缓解，其主动脉根部斑块面积显著减小。而且巨噬细胞CGI-58过表达引起血浆总胆固醇和高密度脂蛋白（HDL）胆固醇水平上调。同时CGI-58 Tg/ApoE−/−小鼠来源的腹腔巨噬细胞胆固醇逆向转运相关蛋白（包括PPARa, PPARγ, LXRα, ABCA1, 和ABCG1）表达水平要比其CGI-58 WT/ApoE−/−对照小鼠来源的巨噬细胞明显增高，而且巨噬细胞流向HDL和脂蛋白A1（Apo A1）的胆固醇流量在CGI-58 Tg/ApoE−/−小鼠也要明显增加。对于Mac-CGI-58 Tg小鼠实验发现，该小鼠血清炎症介质（如TNF-α和IL-6）水平比其对照的Mac-CGI-58 WT明显降低，巨噬细胞线粒体功能更加正常。这些现象在对RAW264.7细胞CGI-58基因敲除后给予LPS刺激实验中也得到了反向验证。更有意义的是，CGI-58在巨噬细胞过表达和沉默后分别诱导/促进和抑制PPARγ的表达及其活性。更有甚者，PP