肝细胞外泌体miR27a下调Ubc13蛋白诱导肝纤维化的分子机制研究

The regulatory role of cellular contacter-exosomes in liver fibrosis is not clear. In our previous studies, hepatocytes (HCs) simulated by promoting fibrosis factors could release more amount of exosomes with up-regulated miR27a. These exosomes would inhibit Ubc13 proteins synthegenesis of target cells including HCs and hepatic stellate cells (HSCs), aggravate liver inflammation and fibrosis, and impair mitochondria function of target cells. Then we explore the mechanisms of overexpression or suppression exosomal genes in miR27a-Ubc13 axis that regulate its downstream target signaling pathway in liver fibrosis and impact on inflammation of HCs, activation of HSCs and mitochondria function of target cells in vitro. We also clarify the rules of overexpression exosomal miR27a or knockout Ubc13 that exacerbate liver fibrosis in liver-specific knockout Ubc13 mice and liver fibrosis mice induced by CCl4, DEN or high fat diet. Finally, the expression of serum exosomal genes and their association with prognosis were observed in patients with liver fibrosis caused by chronic hepatitis B or nonalcoholic steatohepatitis. The results of the study will help us to explore the molecular mechanisms of exosomes in liver fibrosis and provides a new theoretical and experimental basis for the clinical treatment of liver fibrosis.

细胞“联络员”外泌体在肝纤维化中的作用机制尚不清楚。我们发现促肝纤维化因子刺激肝细胞（HC）产生富含miR27a的外泌体，抑制调控靶细胞[HC和肝星状细胞（HSC）]的Ubc13蛋白合成而加重肝炎、肝纤维化和靶细胞线粒体功能损害。本课题拟从细胞器外泌体水平用siRNA技术过表达或抑制干扰miR27a-Ubc13调控轴基因，探索其对HC炎症、HSC活化、靶细胞线粒体损害等生物学功能和下游肝纤维化靶通路的影响。并构建Floxed基因法肝脏特异性Ubc13敲除鼠和CCl4、DEN、高脂饮食肝纤维化小鼠模型，从体内角度阐述过表达外泌体miR27a或Ubc13缺失加剧肝纤维化的作用。最后观察在慢性乙肝和非酒精性脂肪性肝炎肝纤维化人群中血清外泌体这些基因的表达及与预后的关联。研究结果将有助于从外泌体这一新视角探索肝纤维化分子机制，为临床治疗肝纤维化提供新的理论和实验依据。

【目的】外泌体介导的线粒体自噬在代谢相关性脂肪肝疾病 (MAFLD) 中肝细胞 (HC) 和肝星状细胞 (HSC) 之间的串扰中的作用仍然未知。【方法】通过超速离心、外泌体标志蛋白、纳米粒子跟踪（NTA）以及PKH26荧光标志等鉴定肝脏外泌体。我们通过miRNA/mRNA芯片、荧光素酶报告及PCR等方法筛选出外泌体miR27a-PINK1调控轴。在体外实验中，过表达或抑制表达外泌体miR27a-PINK1调控轴基因， 通过免疫荧光（IF）、mtSOX、 mtCMXRos、OCR、PCR、Western blot等检测对HSCs活化增殖及线粒体功能的损害。在体内实验中，构建高脂饮食（HFD/MCD）、四氯化碳（CCl4）诱导的MAFLD肝纤维化小鼠模型，HE、油红和天狼星红染色判断肝组织病理变化，电镜、IF、PCR及Western blot等观察外泌体miR27a或PINK1缺失对肝纤维化的作用。【结果】在MAFLD 患者和小鼠中，血清外泌体 miR-27a水平显著升高，与肝纤维化程度正相关。外泌体miR-27a从脂毒性HCs中释放出来，并特异性地传递给受体-活化的HSCs。 PINK1是miR-27a的关键靶基因，主要介导线粒体自噬。HCs中过表达miR-27a或抑制PINK1或脂毒性外泌体miR-27a会损害LX2中的线粒体功能（抑制线粒体自噬、呼吸、膜电位和转录，同时促进ROS产生），并刺激HSC分化为成纤维细胞（促进活化和增殖，抑制自噬）。高外泌体miR-27a血清水平和缺乏肝脏 PINK1介导的线粒体自噬与 MAFLD 小鼠的肝纤维化直接相关。脂毒性HC外泌体移植加重了肝硬化MAFLD小鼠肝脏中PINK1介导的线粒体自噬抑制、脂肪性肝炎、脂质沉积和纤维化的程度。无论是在体外还是在体内，来自miR-27a 敲低HCs的外泌体都不能产生上述恶化作用。【结论】脂毒性HC分泌的外泌体优先被活化的HSC吸收。外泌体miR27a负向调节肝脏PINK1介导的线粒体自噬，可增强HSC活化和增殖，从而加速MAFLD肝纤维化。这些结果表明，抑制HC-外泌体miR-27a表达可用作 MAFLD肝纤维化的潜在诊断标志物和治疗靶点。