【作者】 李彬；

【导师】 陈小冬；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 成纤维细胞生长因子21(fibroblast growth factor 21,FGF21)是FGF家族中的一员,它能够抵制肥胖,增强胰岛素敏感性以及改善各种代谢疾病。我们前期在肝细胞中的研究发现,FGF21对脂质积累、脂质引起的内质网应激、线粒体功能和代谢性炎症等都有重要的控制作用。同时,我们也发现FGF21受到转录因子NFkB、CEBPs和PPARα的转录水平调控以及microRNA26对它翻译水平的调控。自噬是细胞的一种自我保护机制。目前研究颇热的是自噬对细胞脂肪代谢及相关疾病的影响,脂噬也已成为自噬的一个重要分支。因此我们假设FGF21与自噬之间存在着紧密的联系,本研究在证实这种联系的基础上,进一步探究FGF21影响自噬的分子机制。目前我们主要探索FGF21调控自噬的机制。采用超表达FGF21,敲减FGF21,PI3K/AKT抑制剂LY294002以及AMPK激活剂AICAR等技术手段对细胞进行处理,并通过定量PCR,Western Blot,MDC染色等实验方法检测细胞的自噬相关基因mRNA和蛋白水平变化以及细胞形态学上的变化,进而探索FGF21在HepG2细胞中对自噬的调控机理。具体结果如下:1.PA会显著上调自噬相关基因的mRNA水平、蛋白水平以及形态学水平,并且会显著增强FGF21的mRNA的水平。2.超表达FGF21,自噬相关基因的mRNA水平以及蛋白水平会显著上调,MDC染色中出现的强度自噬也印证了此结论。此外,Beclin-1的蛋白水平也上调。3.与超表达相反的是,敲减FGF21,自噬相关基因的mRNA水平以及蛋白水平显著下调,MDC染色中自噬的强度也减弱。此外,Beclin-1的蛋白水平也显著下调。4.对HepG2细胞,分别用AMPK的激活剂AICAR、P13K/AKT的磷酸化抑制剂LY294002以及FGF21的重组蛋白处理,发现AICAR、LY294002以及FGF21的重组蛋白均能显著增强自噬相关基因的蛋白水平以及形态学水平,并且使FGF21的以及AKT的总蛋白水平显著降低。5.敲减FGF21后使用AICAR、LY294002以及FGF21的重组蛋白刺激,使用MDC染色检测自噬的形态学水平,其中LY294002以及FGF21重组蛋白在FGF21敲减实验后的回复作用最显著,因此我们推测FGF21可能是通过PI3K/AKT途径来调控自噬。6.我们之前的研究发现NFκB/p65能直接结合到FGF21的DNA启动子上并调控FGF21的转录。本研究中,超表达p65后,能显著降低自噬的蛋白水平,而且AKT总蛋白水平显著增强。因此,p65极有可能是通过FGF21来调控HepG2的细胞自噬。综上所述,本研究的结论是:PA可以增强HepG2细胞的自噬水平,此外FGF21是通过PI3K/AKT途径来调控自噬,而p65极有可能是通过FGF21进而调控该细胞的自噬水平。更多还原

【Abstract】 Fibroblast growth factor 21(fibroblast growth factor 21,FGF21),a member of FGF family,can resist obesity,enhance insulin sensitivity and improve various metabolic diseases.Previous studies in hepatocyte revealed that FGF21 plays important roles in regulating lipid accumulation,lipid-induced ER stress,mitochondrial dysfunction and metabolism-related inflammation.Simultaneously we found that FGF21 transcription is regulated by transcription factors NFκB,CEBPs and PPARα,while microRNA26 can control FGF21 translation.Autophagy is a self-protection mechanism which is extensively studied in lipid metabolism and lipid-related diseases,and currently lipophagy is thought as a new branch of autophagy.Therefore,in this study,we hypothesized that there exists some relationship between FGF21 and autophagy.Thus,based on confirming this relationship,we explored the potential molecular mechanism that FGF21 influenced autophage in HepG2 cells.In the present studies,to detect the mechanism of FGF21-regulating autophagy,we overexpressed or knocked down FGF21 in HepG2 cells,or treated cells with AKT inhibitor LY294002 or AMPK activator AICAR or FGF21 recombinant protein,then detected the mRNA and protein levels of the biomarker genes and observed the change of autophagy morphology though real-time PCR,Western Blot and MDC dyeing etc.methods The acquired detail results are as follows:1.PA significantly up-regulated the mRNA and protein levels of autophagy key genes and changed the morphology of autophagosome,and simultaneously remarkably enhanced the level of FGF21 mRNA.2.Overexpressed FGF21 can significantly enhanced the mRNA and protein levels of autophagy key genes,and the stronger autophagy was observed by MDC dyeing in FGF21-overexpressed HepG2 cells compared to control.3.By contrast,FGF21 knockdown conspicuously reduced the mRNA and protein levels of autophagy key genes,and the cell autophagy also became weaker by MDC dyeing compared to control.4.In HepG2 cells treated with AMPK activator AICARor PI3K/AKT phosphorylation inhibitor LY294002 or FGF21 recombinant protein,we found that the mRNA and protein levels of autophagy key genes and morphological level were increased in all the treated groups accompanied by the remarkable decreases of FGF21 and total AKT protein levels.5.By contrast,In HepG2 cells with AICAR,LY294002 or FGF21 recombinant protein treatment,FGF21 knockdown returned the effect of LY294002 and FGF21 recombinant protein but not AICAR in autophagy morphological level.Therefore,we deduced that FGF21 regulates autophagy through PI3K/AKT pathway.6.Our previous study found NFkB/p65 can directly bind to FGF21 promoter DNA and regulate FGF21 transcription.Here,p65 overexpression significant enhanced the protein levels of autophagy key genes and the level of total AKT protein.Therefore,autophagy possibly is regulated via p65-FGF21-PI3K/AKT pathway in HepG2 cells.In summary,the conclusions of this study are:PA can up-regulate autophagy and autophagy possibly is regulated via p65-FGF21-PI3K/AKT pathway in HepG2 cells.更多还原