MYO5B突变影响肝细胞胆盐外运泵的机制及其与肝脏表型关系

胆盐外运泵(BSEP)的原发功能缺陷导致严重胆汁淤积症。我课题组新鉴定MYO5B突变致肝内胆汁淤积谱系(Hepatology 2017)，不伴经典腹泻表型；临床、病理、胆汁酸谱的改变与原发BSEP缺陷病极为相似，提示继发BSEP功能障碍可能是引起胆汁淤积的机制。目前认为MYO5B-RAB11A相互作用为BSEP重定位所必需，但无法完美解释胆汁淤积原因和不同突变的表型差异机制。由于MYO5B在肝细胞中的功能仍不清楚，本研究拟通过构建MYO5B体内外敲低/敲除模型及挽救实验，应用活细胞显像、组织化学、RNA-seq、定量蛋白组学等探明MYO5B在肝细胞中具体功能及MYO5B缺陷影响肝细胞BSEP的具体机制；通过体外肝/肠上皮细胞MYO5B敲除模型，分别转染不同表型特有的MYO5B突变质粒，探究不同MYO5B突变引起不同表型的机制，为MYO5B突变及其它原因胆汁淤积症的治疗研究提供理论依据。

Primary bile salt export pump (BSEP) would lead to severe cholestasis. Our recent study newly found that MYO5B defect causes a full spectrum of isolated intrahepatic cholestasis, without classical phenotype of diarrhea (Hepatology, 2017). Its similarity to primary BSEP defect in changes of clinical presentation, liver pathology and bile acid profiling suggested secondary BSEP dysfunction. The re-location of BSEP requires MYO5B-RAB11A interaction but such theory couldn’t perfectly explain the BSEP abnormality and the phenotype difference. It is still unclear about MYO5B function within hepatocyte, so in this study, we plan to construct in vitro and in vivo MYO5B hepatocyte knockdown/knockout models & related rescue experiments, and by live cell imaging, RNA-seq, quantitive proteinomics etc. we try to elucidate the MYO5B function within hepatocytes and how its mutants affect the BSEP life cycle; we also plan to construct stable MYO5B knockout hepatocyte/enterocyte models in vitro, and by transfecting MYO5B plasmid and mutated MYO5B plasmids from both phenotype separately, we try to explain how different mutations cause phenotype difference. Such findings will provide clues for precise treatment in MYO5B defects and other cholestasis.