Rs1044925单核苷酸多态性对动脉粥样硬化中巨噬细胞脂质代谢影响的研究

Once taking up enough lipid, the macrophage will turn into foam cell, which is closely related to coronary artery disease (CAD) as well as atherosclerosis, the pathological basis of CAD. Acyl-coenzyme A:cholesterol acyltransferase (ACAT1), coded by Soat1 gene, is the dominant coenzyme esterifying free cholesterol (FC) to cholesterol ester (CE). Previous studies show the association between Soat1 gene Single Nucleotide Polymorphism (SNP) rs1044925 A to C mutation and lipid profile in circulation, as well as CAD risk. The relationship and mechanism, however, remains to be confirmed and illustrated. This research aims to: 1. Ascertain the characteristics and functional pathway of rs1044925 via bioinformatics; 2. Use CRISPR/Cas9 modifying rs1044925 SNP from A to C in embryonic stem cell, which will be differentiated to macrophage; 3. Find the influence of rs1044925 to lipid metabolism in macrophage and the underlying mechanism by cellular test such as cholesterol mass assay. Our research is expected to further illuminate the relationship between rs1044925 and lipid metabolism, as well as the mechanism, and provide new theoretical base and experimental evidence for prevention and therapy of atherosclerosis.

冠心病及其病理基础动脉粥样硬化与巨噬细胞摄取脂质转化为泡沫细胞密切相关，巨噬细胞内调控游离胆固醇转化为胆固醇酯的主要同工酶为Soat1基因编码的ACAT1酶。既往研究发现Soat1基因rs1044925单核苷酸多态性A至C的突变对人体循环内脂质水平和冠心病发病风险存在关联，但该相关性和其机制有待进一步确定和阐明。本课题拟：1.通过生物信息学调查Soat1基因rs1044925相关信息，明确其基本属性和工作通路；2.通过CRISPR/Cas9构建rs1044925A至C改造的干细胞并进一步将其分化为巨噬细胞模型；3.通过胆固醇质量检测等细胞实验及相关指标检测明确rs1044925对巨噬细胞脂质代谢影响及其机制。该研究有望进一步阐明rs1044925与血脂代谢的相关性及其工作机理，为动脉粥样硬化的防治提供新的理论基础和实验依据。

冠心病及其病理基础动脉粥样硬化与巨噬细胞摄取脂质转化为泡沫细胞密切相关，巨噬细胞内调控游离胆固醇转化为胆固醇酯的主要同工酶为Soat1基因编码的ACAT1酶。本课题建立AKRxDBA/2杂交小鼠群组并进行QTL定位，用生物信息学探究候选基因Soat1基因在两家系间的差异；使用CRISPR/Cas9技术编辑DBA/2干细胞Soat1基因，并定向分化为巨噬细胞，进一步基因编辑后进行巨噬细胞功能和脂质代谢检测。同时在本课题的资助下，研究了pH敏感的pHrodo探针在动脉粥样硬化疾病诊断中的作用及机制：完成了使用pHrodo探针处理动脉粥样硬化标本，应用分子生物学手段检测pHrodo探针作用于动脉粥样硬化病变酸性区域的具体机制；制造NHe1缺乏小鼠，观察NHe1缺乏对动脉粥样硬化是否具有抑制作用，观察病变区域内局部酸化是否有影响；将Nhe1或IgE受体FcεR1缺陷小鼠的骨髓抑制于Apoe-/-小鼠，观察其动脉粥样硬化病变程度，并检测通过对巨噬细胞的影响导致的病变程度变化的机制；静脉注射近红外荧光pH敏感探针LS662，然后进行荧光分子计算机断层扫描成像，明确了其可用于识别活体小鼠动脉粥样硬化病变中的酸性区域的作用；项目同时研究了急性心肌梗死恢复期患者与健康人循环外泌体对心肌细胞、内皮细胞的氧化损伤修复的作用。研究健康人循环外泌体通过miR-342-3p靶向SOX6、TFEB细胞凋亡与自噬调控心肌细胞损伤修复；miR-193a-5p通过调控ACVR1参与内皮损伤修复。本项目进一步阐明了动脉硬化发生发展的病理生理过程及其机制，为动脉粥样硬化的诊断、治疗提供了新的理论基础和实验依据。