NS2TP在非酒精性脂肪性肝病中保护线粒体功能的机制研究

The mechanism of self-protection in hepatocytes with NAFLD still needs to be clarified. It is a good starting point to analysis the factors of protecting mitochondrial function. NS2TP can inhibit mitochondrion damage and apoptosis, and bind with deacetylase Sirt1 as well. Our previous study showed that increased expression of NS2TP in liver tissues of mouse NAFLD models could be induced by high fatty diets, and levels of NS2TP increased when Sirt1 kept silence in HepG2 cell lines. Here we have a hypothesis that NS2TP should protect mitochondrion function in the steatosis stage and the protein level of NS2TP might be regulated by Sirt1. These experiments will performed on mouse NAFLD models of high fatty diet, steatosis cell models in vitro or hepatoma cell lines. And this project will be carried out by using immunofluorescence, electronmicroscope, mitochondrial function analysis, flow cytometry, Western blot, co-immunoprecipitation, immunoprecipitation and RNAi. This study is to explore the expression and sublocalization of NS2TP in adipose degeneration hepatocyte mitochondrion, analyze the effects and mechanisms of NS2TP on apoptosis and mitochondrial function, and further investigate the deeply mechanism of NS2TP regulated by Sirt1 at protein level. This project is expected to supply a further thinking for the self-protection function of hepatocyte, and also be a novel idea for the NAFLD research.

非酒精性脂肪性肝病（NAFLD）涉及的肝细胞自我保护机制尚未完全阐明，线粒体功能保护因素分析是重要着眼点。NS2TP能抑制非肝细胞线粒体损伤和细胞凋亡，还能与去乙酰化酶Sirt1结合。我们发现Sirt1沉默后NS2TP蛋白水平增高、高脂饮食诱导NAFLD小鼠肝组织NS2TP表达增多而Sirt1减少。因此提出“NS2TP保护脂肪变性肝细胞线粒体功能，NS2TP蛋白水平受Sirt1调控”假说。本项目拟利用NAFLD小鼠模型、体外细胞模型和HepG2细胞系，通过免疫荧光、电子显微镜、线粒体功能分析、流式细胞术、Western blot、免疫（共）沉淀和RNAi等技术，观察NS2TP在脂肪变性肝细胞线粒体中的表达和定位，分析NS2TP对脂肪变性肝细胞凋亡和线粒体功能的影响及机制，探讨Sirt1介导的NS2TP蛋白表达增高的机理。本项目将为阐释肝细胞自我保护机制和NAFLD发病机制研究提供新切入点。

非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）依据病理学分期分为非酒精性单纯性脂肪肝、非酒精性脂肪性肝炎（non-alcoholic steatohepatitis，NASH）、NASH相关肝纤维化和肝硬化等阶段。NAFLD已成为全球重要的公共健康问题之一。如何阻止病程进入NASH及后续阶段是当前临床需要重点关注和解决的问题。研究表明，线粒体功能障碍是促进非酒精性脂肪性肝病（NAFLD）进展的关键原因。NS2TP是我们项目组长期给予关注的功能基因。NS2TP在神经系统疾病中的功能已有较多的深入研究。根据文献报道及我们前期的研究结果，我们提出“NS2TP增高是脂肪变性肝细胞自我保护线粒体功能的表现”。①本项目制备了兔抗小鼠NS2TP（亦名CHCHD2）多克隆抗体，解决了重要试剂瓶颈，为后续动物模型研究提供了重要基础。克隆了C57BL/6小鼠NS2TP基因表达序列，构建了重组原核表达载体pET- 28a(+)-mCHCHD2，大量诱导后获得了纯化重组蛋白，制备了高效价的兔抗小鼠CHCHD2多克隆抗体。②本项目还初步明确了NS2TP抑制肝细胞凋亡、发挥NAFLD中肝细胞保护作用的可能机制。构建了小鼠NASH模型和体外脂肪肝细胞模型；观察了NASH患者肝组织切片、模型小鼠肝组织和体外模型细胞中NS2TP 的表达、分布情况，观察了氧化应激对NS2TP亚细胞定位的影响。观察了模型小鼠肝组织凋亡水平及与NS2TP表达水平的关系。初步证实了NS2TP通过调控PI3K/Akt信号通路抑制肝细胞凋亡，发挥了肝细胞保护功能的可能机制。为进一步阐释肝细胞“自我保护”机制提供新的切入点，为NAFLD发病机制研究提供新的思路。