Sam68调控巨噬细胞脂噬促进钙化性主动脉瓣疾病的机制研究

Calcific aortic valve disease (CAVD) is the most common heart valve disease in the world. Clinical specimen analysis suggested that improving macrophage lipid metabolism helps to prevent CAVD, and macrophage lipophagy deficiency may be closely related to the progression. Sam68 is one of the members of the intracellular signal transduction and RNA activator family. The preliminary data of our group found that Sam68 negatively modulates autophagy and the down-regulation of Sam68 helps to enhance macrophage lipophagy. This present project will focus on the association of Sam68 and macrophage lipophagy in CAVD, and plans to do experiments like: (1) through down-regulating the expression of Sam68 by RNA interference and gene editing techniques, to clarify the effects of Sam68 negative modulation and fusion impediment on autophagy, and to elucidate the molecular mechanism of Sam68 suppressing macrophage autophagy by determining location and targeting proteins; (2) through macrophage lipophagy model stimulated by ox-LDL in vitro, to establish the effects of Sam68 down-regulation on cholesterol efflux, foam-like cell formation, pro-inflammatory factors release, apoptosis, protein degradation and efferocytosis, and to investigate the effects of macrophage lipophagy on calcification related proteins produced from valvular interstitial cells in three dimensional co-culture system; (3) through producing tissue-specific double knockout mice by Sam68 myeloid-specific knockout mice mating with ApoE knockout mice, to demonstrate the effects of inhibiting Sam68 to reduce heart valve calcification by the CAVD model arising from high-fat diet and the rescue model from bone marrow transplantation, and to emphasize the protective role of macrophage lipophagy for CAVD prevention and treatment combined with clinical data.

钙化性主动脉瓣疾病（CAVD）是全球最常见心脏瓣膜疾病。临床研究提示，改善巨噬细胞脂代谢减轻瓣膜钙化，巨噬细胞脂噬不足与CAVD发病相关。Sam68是细胞内信号转导及RNA激活蛋白家族成员之一，课题组前期首次发现下调Sam68增强巨噬细胞脂噬。本项目拟：⑴利用RNAi和基因编辑技术下调Sam68表达，明确Sam68负性调节自噬及阻碍融合的效果，探讨靶点以阐明分子机制；⑵对下调Sam68巨噬细胞予ox-LDL刺激，观察加速自噬流对巨噬细胞脂质外排、泡沫样细胞形成、促炎因子释放、凋亡、蛋白降解、胞葬影响，并利用共培养体系，观察巨噬细胞脂噬对继发瓣膜间质细胞钙化影响；⑶将Sam68髓系特异性敲除小鼠与ApoE敲除小鼠杂交产生组织特异性双敲小鼠，联合骨髓移植和高脂喂养以建立在体模型，观察下调Sam68减轻瓣膜钙化效果，结合人体标本分析以凸显巨噬细胞脂噬的保护性作用，建立CAVD防治新靶点。

钙化性主动脉瓣疾病（CAVD）是目前全球心血管负担前三名疾病之一。Sam68是细胞内一种参与信号转导且具有RNA结合活性的蛋白。前期发现下调Sam68增强巨噬细胞自噬和脂代谢。本项目在此基础上，以巨噬细胞和瓣膜间质细胞为核心，多维度探讨Sam68调节脂代谢、自噬和成骨分化，继而促进CAVD作用机制。主要研究内容包括：① 通过人体标本检测，发现在瓣膜钙化过程中存在Sam68表达上调、自噬流受阻，脂质沉积；② 体外TNF-α刺激人瓣膜间质细胞钙化，证明Sam68通过与STAT3互作，调节后者磷酸化水平，促进瓣膜间质细胞成骨分化，阻碍自噬流；③ 体外ox-LDL刺激小鼠巨噬细胞，显示下调Sam68后脂代谢加速，泡沫细胞形成减少，同时巨噬细胞促炎因子表达减少，向M2亚型极化增加；④ 将Sam68髓系特异性敲除小鼠与ApoE敲除小鼠杂交，繁育组织特异性双敲小鼠，建立高脂喂养CAVD在体模型，验证Sam68调节巨噬细胞功能，进而影响瓣膜钙化。以上研究为CAVD病理生理研究提供新的证据，有助从巨噬细胞脂代谢角度为临床防治找到新的靶点。