非酒精性脂肪性肝病中肝细胞分泌的胞外小体miRNA对库普弗细胞的调节作用的研究

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide. It begins with simple hepatic steatosis, and can progress to steatohepatitis, fibrosis and even cirrhosis. Although genetic factor, overweight, insulin resistance, an altered dietary pattern and gut microbiota are all identified as key risk factors, the knowledge about the molecular mechanisms involved in disease onset and progression is still limited. Kupffer cells, the liver macrophages, are found to play an essential regulatory role in NAFLD. Yet the crosstalk between hepatocytes and Kupffer cells remains unclear. In NAFLD, the signature expression pattern of hepatocyte-derived exosomal miRNAs has been appreciated in disease diagnosis and prognosis. Yet the biological functions of these extracellular miRNAs remain to be characterized. Recently in alcoholic liver disease, a regulatory role of a hepatocyte-derived exosomal miRNA in monocyte/macrophage activation upon LPS stimulation was reported. Here, we hypothesize that hepatocyte-derived exosomal miRNAs can regulate the function of Kupffer cells in NAFLD. Expression pattern of hepatocyte-derived exosomal miRNAs will be profiled through microarray analysis using an in vitro model of NAFLD. Whether hepatocyte-derived exosomes are received by Kupffer cell will be visualized following fluorescent labeling of exosomes. Potential function of exosomal miRNAs in Kupffer cells will be examined by overexpression and knockdown experiments. Downstream targets of exosomal miRNAs will be verified following computational prediction. This study will not only elucidate the biological function of hepatocyte-derived exosomal miRNAs in NAFLD, but also advance our knowledge about hepetocyte-Kupffer cell crosstalk during NAFLD progression.

非酒精性脂肪性肝病（NAFLD）是除酒精和其他明确损肝因素外导致的肝细胞内脂肪过度沉积为主要特征的临床病理综合征，包括单纯性脂肪肝、非酒精性脂肪性肝炎及其相关肝纤维化和肝硬化。库普弗细胞（KC）在NAFLD的发展过程中具有关键作用，但其与肝细胞的互作还有待深入研究。NAFLD中肝细胞分泌的胞外小体miRNA的表达谱对其诊断和预后有意义，但胞外miRNA的生物学功能尚不明确。最近在酒精性肝病中发现，肝细胞胞外小体来源的miRNA可以降低单核细胞和巨噬细胞对脂多糖刺激的敏感性。本项目拟探求NAFLD中肝细胞分泌的胞外小体miRNA对KC功能的影响。利用体外细胞模型，先用基因芯片技术检测肝细胞外泌miRNA的表达谱，并通过荧光标记观察胞外小体是否进入KC，再通过过量表达和基因沉默实验探究miRNA对KC的调节作用，最后验证miRNA的靶基因。本项目不仅将有助于理解NAFLD中肝细胞分泌的胞外小体miRNA的功能、肝细胞与KC的互作，且有望为NAFLD的治疗和药物研发带来新的启示。

非酒精性脂肪性肝病（NAFLD）是指除酒精和其他明确的损肝因素外所致的肝细胞内脂肪过度沉积为主要特征的临床病理综合征，现已成为欧美等发达国家和我国富裕地区慢性肝病的重要病因。库普弗细胞（KC）做为肝脏中定居的巨噬细胞，在NAFLD的发展过程中具有关键作用，但其与肝细胞的互作还有待深入研究。本项目通过游离脂肪酸诱导肝细胞建立NAFLD的体外模型，探究NAFLD肝细胞分泌的胞外小体miRNA对环境中巨噬细胞功能的调节作用。我们通过实时荧光定量PCR法证实miR-192等miRNA在脂肪病变肝细胞来源的胞外小体中富集表达，通过激光共聚焦显微镜观察发现荧光标记的胞外小体可以进入环境中的巨噬细胞。我们发现外源性的miR-192进入巨噬细胞后可抑制LPS诱导的M1型极化和炎性细胞因子的产生，而促进巨噬细胞的M2型极化及抑炎细胞因子的产生。我们进一步通过双萤光素酶报告基因实验证明IL6ST是miR-192的下游直接作用基因，miR-192对巨噬细胞极化的调节作用具有IL6ST依赖性。因此我们的研究结果提示，NAFLD肝细胞分泌的miR-192可以进入周围环境中的巨噬细胞，并通过直接抑制其靶基因IL6ST，调节巨噬细胞的极化，抑制炎症反应的发生。这可能是NAFLD在单纯性脂肪肝-非酒精性脂肪性肝炎-肝纤维化-肝硬化-肝癌这一病程发展的初级阶段，机体产生的自稳保护性调节机制。