【作者】 张艳；

【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， 2011， 硕士

【摘要】 肝脏脂肪过度积累会导致肝脏脂肪变性,脂肪肝,肝癌等一系列肝脏疾病。FGF21是近几年发现的对脂代谢疾病有重要作用的细胞因子,其在肝脏中表达丰度很高,本文利用HepG2细胞系作为肝细胞模型,通过油红O染色确定了FGF21超表达对肝细胞甘油三酯积累的影响,分析了其超表达对肝细胞中糖脂代谢相关基因的转录表达水平的调控,并通过Western Blot和Real-time PCR方法进一步分析了过量表达的FGF21对调节脂肪酸合成的关键转录因子SREBP1c的表达、加工的影响；最后借助双荧光素酶报告基因检测系统分析了SREBP-1c对FGF21启动子转录活性的影响。研究结果证明FGF21通过抑制SREBP1c的表达和加工成熟过程,从而减少SREBP1c靶基因即脂肪酸合成相关基因的转录,最后达到减少肝细胞甘油三酯积累的目的。另外本文还克隆猪FGF21基因,并对其进行组织表达分析；原核表达并初步纯化该基因的蛋白质,以猪为模型,为研究该基因的功能提供基础平台。以下为本文的具体研究结果：1.FGF21抑制肝细胞的脂肪酸合成过程,但不影响脂肪酸的吸收和VLDL的组装,进而使肝细胞内的甘油三酯积累减少；FGF21也能够挽救由高浓度脂肪酸引起的SREBP-1c和FAS表达量的增加；FGF21抑制SREBP-1c基因的转录和蛋白质的加工及入核；SREBP-1c能够抑制FGF21的启动子活性,形成一个反馈抑制的调节环路；最后FGF21抑制肝细胞的脂肪酸氧化及线粒体的生物合成。另外葡萄糖和ChREBP能够上调FGF21 mRNA的表达水平,而游离脂肪酸和S(?)REBP-1c则抑制FGF21的转录表达。2.FGF21在糖代谢中几乎不发挥作用。只是在基础条件下抑制糖异生的关键酶PEPCK基因的转录。3.获得猪FGF21全编码区627 bp,共编码208个氨基酸；该基因包含3个外显子和2个内含子,其基因组结构及各个外显子长度与人和小鼠FGF21的基因组结构相似；FGF21在猪各组织中广泛表达,其中在肝脏,各脂肪组织(包括肠系膜脂肪组织,附睾脂肪组织和皮下脂肪组织)及肾脏等代谢器官中表达量较高。更多还原

【Abstract】 The access accumulation of triglyceride in hepatocytes may lead to a series of hepatic diseases, such as steatosis, fatty liver, heppatoma. FGF21 is a recently discovered cytokine playing an important role in lipid metabolism. In this study, HepG2 cell line was applied to conduct the research of FGF21 on lipid metabolism in atocytes in vitro. Firstly, the alteration of triglyceride content in hepatocytes after FGF21 overexpression was measured by oil O staining. Then the transcriptional expressions of genes involved in lipid metabolism in hepatocyte were assessed after over-expression FGF21. Further western blotting, immunocytochemistry and Real-time PCR were used to detect the expression, processing and entering into nucleus of SREBP-lc, a key transcriptional factor participated in lipogenesis. At last, we evaluated the regulation of SREBP-1c on FGF21 promoter using dual-luciferase reporter assay system. Our study manifested that FGF21 inhibited the transcriptional expression and processing of SREBPlc, and then reduced the mRNA of SREBPlc target genes, and at the end, reduced TG accumulation in HepG2 cells, In addition, using Meishan pig as a research object, we cloned the procine FGF21 and analysed the profiles of its expressions in various tissues for the first time. And then the FGF21 protein was expressed in E. coli BL21 (DE3) and was roughly purified. The detailed results are as follows:1. Genes participated in fatty acids de novo synthesis and esterification was down-regulated by FGF21. However, it has no significant effect on absorption of fatty acids and VLDL assembling. Oil O staining manifests that cellular triglyceride content was suppressed because of FGF21 over-expression. FGF21 protects hepatocytes from the up-regμlation of FAS and SREBP-1c causing by free fatty acids. FGF21 can inhibit the transcriptional expression, the processing and the entering into nucleus of SREBP-lc, and SREBP-1c in turn suppresses the promoter activity of FGF21, forming a feedback inhibition loop. FGF21 elicits the down-regulation of fatty acids oxidation. Glucose and ChREBP could escalate FGF21 mRNA level depending on dosage and time, and in verse fatty acids and SREBP-1c can suppress the transcription level of FGF21 in hepatocytes, also depending on dosage and time.2. FGF21 has little effect on hepatic glucose metabolism, only inhibiting gluconeogenesis in normal condition to some extent.3. The coding region of porcine FGF21 and partial non-coding sequence of porcine FGF21was amplified and then bioinformatic analysis was conducted. The coding region of porcine FGF21covers 627bp, encoding 208 amino acids. There are three exons and two introns in the genomic sequence of FGF21, and this is similar with human and mice’s FGF21 genomic structure. FGF21 expresses in various tissues of pig, and its expression is abundant in liver and adipose tissue. This implies the possible participation of FGF21 in metabolism.更多还原