PNPLA3基因在NAFLD肝细胞脂滴和脂肪代谢中的作用及机制研究

Nonalcoholic fatty liver disease (NAFLD) is a common disease, and its hereditary factors have attracted more and more attention. The typical pathological feature of liver steatosis is the lipid deposition in liver cells in the form of lipid droplets (LDs).In recent years, PNPLA3 polymorphism has become a hot topic in research of NAFLD, and associated with liver inflammation development and disease progression. To examine the role of PNPLA3 in triglyceride metabolism and elucidate the mechanism by which the I148M substitution affects liver lipid content, we delivered human PNPLA3 cDNA, PNPLA3-I148M and siRNA-PNPLA3 in adenovirus vector, which are transferred to primary liver cells or Huh-7 cells. Then the factor of lipid droplets metabolism were detected in each cell group. We also indentify the PNPLA3-interacting protein-OXPAT and facilitate the illustration of the molecular mechanism of PNPLA3. Adenoviral vectors are transferred to rat model of NAFLD. Then the blood biochemical parameters and lipid metabolism of liver were measured, and the hepatic steatosis was evaluated by immunohistochemical analysis to verify the relationship between the PNPLA3-I148M and development of NAFLD. The research explores the molecular mechanisms and provides strong theoretical basis and technical support of clinical treatment to NAFLD.

非酒精性脂肪肝是一种常见肝脏疾病，其典型病理变化是肝细胞内过多甘油三酯沉积，并出现脂滴。目前遗传因素导致的NAFLD逐渐引人关注，其中PNPLA3 rs738409基因多态性经实验证实与肝内脂肪代谢密切相关。本项目拟通过制备PNPLA3-WT及其突变体PNPLA3-I148M基因或si-PNPLA3的腺病毒表达系统，感染原代肝细胞或建立稳转的Huh-7细胞，检测各组肝脏细胞中脂滴代谢相关因素的差异，分析PNPLA3与脂滴蛋白OXPAT的相互作用，探究PNPLA3在脂滴脂肪代谢中的作用，以及PNPLA3-I148M影响脂滴脂肪蓄积的分子机制。体内实验通过建立NAFLD大鼠模型，腺病毒介导PNPLA3-WT及PNPLA3-I148M基因在肝脏中过表达或沉默表达，检测血清及肝脏中各脂肪代谢指标，分析肝脏脂肪病变情况，体内验证PNPLA3-WT及PNPLA3-I148M与NAFLD发生发展的关系。

非酒精性脂肪性肝病(NAFLD)在我国是继病毒性肝炎之后第二位常见肝病，常伴发肥胖、2型糖尿病和高脂血症等代谢紊乱，是目前成人原因不明肝功异常的最常见原因，并且可以进展至肝硬化甚至肝癌；因此，研究NAFLD的发病机制至关重要。我们之前的研究表明PNPLA3 I148M 基因变异可增加我国NAFLD发生的易感性,并且与肝脏的组织损伤相关,但是具体机制还不清楚,是由于脂肪合成增加还是降解减少所致都没有定论，本研究成功分离了大鼠肝细胞， 并进行了功能鉴定，将用高脂饲料诱导NAFLD大鼠模型,用腺病毒包装SREBP-1c，局部注射进高脂饮食诱导的NAFLD大鼠肝脏中，同时进行体外细胞培养，观察PNPLA3基因表达变化以及甘油三酯以及其中间代谢产物的变化, 同时评价过表达PNPLA3后对胰岛素抵抗的影响，从而探讨PNPLA3引起肝脏脂肪沉积的机制以及可能的信号通路。