【作者】 李钶；

【导师】 杨刚毅；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 博士

【摘要】 第一部分小鼠成纤维细胞生长因子-21 shRNA重组载体的构建目的:构建针对小鼠成纤维细胞因子-21(fibroblast growth factor 21,FGF-21)的短发夹状RNA(short hairpin RNA,shRNA)重组质粒。方法:设计并构建2条针对小鼠FGF-21基因的shRNA反向重复序列,分别克隆至载体pGenesil-1.2的mU6转录启动子下游,构建pGenesil-FGF21-1和2,然后分别与pDsRed-EGFP-FGF-21共转染BHK工具细胞,运用双荧光报告基因验证其有效性,初步筛选出抑制有效的pGenesil-FGF21重组质粒。结果:两pGenesil-FGF21组GFP平均荧光强度/RFP平均荧光强度均低于阴性对照组(均P<0.05),且pGenesil-FGF21-1组最低。结论:成功构建FGF-21-shRNA重组载体,初步筛选出了抑制有效的pGenesil-FGF21重组质粒第二部分成纤维细胞生长因子-21对小鼠3T3-L1脂肪细胞代谢的影响目的:分别建立FGF-21表达缺陷以及FGF-21过表达的脂肪细胞模型,探讨FGF-21表达改变对脂肪细胞糖-脂代谢以及脂肪细胞因子表达的影响。方法:分别将pcDNA-FGF21和pGenesil-FGF21转染3T3-L1脂肪细胞,采用荧光定量PCR和Western Blotting方法检测上述重组质粒对脂肪细胞FGF-21表达的影响,采用2-脱氧-3H-D-葡萄糖(2-deoxy-D-glucose,2-DOG)摄入法测定脂肪细胞葡萄糖转运率(glucose uptake rate,GUR),油红O染色法检测细胞内甘油三酯含量,荧光定量PCR法检测细胞内糖脂代谢相关转录因子的mRNA表达。结果:在脂肪细胞中,pGenesil-FGF21能有效抑制细胞内FGF-21表达(P<0.05),而转染pcDNA-FGF21的脂肪细胞内FGF-21表达显著升高(P<0.05)。FGF-21表达改变时脂肪细胞内以FGFR1改变为主。FGF-21过表达的脂肪细胞GUR显著升高,细胞内TG降低,FGF-21表达缺陷时则相反(P<0.05);葡萄糖转运蛋白-1(glucose transporter-1,GLUT-1)、胰岛素受体底物-1(insulin receptor substrate-1, IRS-1)、过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activated receptor-γ, PPARγ)、过氧化物酶体增殖物激活受体α(peroxisome roliferator-activated receptor-α, PPARα)、脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)、激素敏感性脂肪酶(Hormone sensitive lipase,HSL)和脂肪细胞脂肪酸结合蛋白2 (adipocyte fatty acid-binding protein, ap2)在FGF-21过表达的脂肪细胞内显著增加,FGF-21表达缺陷时降低(均P<0.05);而对葡萄糖转运蛋白-4 (glucose transporter-4,GLUT-4)表达无显著影响。FGF-21过表达的脂肪细胞内Leptin表达降低,FGF-21表达缺陷时Leptin升高(均P<0.05);而FGF-21表达改变对Visfatin及脂联素的表达无显著影响。结论:在脂肪细胞中,FGF-21主要通过betaklotho和FGFR1发挥生物学作用,可能通过GLUT-1和IRS-1途径促进脂肪细胞葡萄糖摄取;并促进脂解作用从而降低细胞内脂质含量;还能调控脂肪因子表达。第三部分成纤维细胞生长因子-21对小鼠Hepa1-6肝细胞代谢的影响目的:分别建立FGF-21表达缺陷以及FGF-21过表达的小鼠肝细胞模型,探讨FGF-21表达改变对肝细胞糖-脂代谢以及重要转录因子表达的影响。方法:分别将pcDNA-FGF21和pGenesil-FGF21转染Hepa1-6肝细胞,采用荧光定量PCR和Western Blotting方法检测上述重组质粒对肝细胞FGF-21表达的影响,采用2-脱氧-3H-D-葡萄糖摄入法测定肝葡萄糖转运率(GUR),荧光定量PCR法检测细胞内糖脂代谢相关转录因子的mRNA表达。结果:pGenesil-FGF21能有效抑制Hepa1-6肝细胞内FGF-21表达(P<0.05),转染pcDNA-FGF21的肝细胞内FGF-21表达显著升高(P<0.05)。betaklotho在过表达FGF-21的肝细胞内呈升高趋势,而在FGF-21缺陷时降低;FGF-21表达改变时肝细胞内以FGFR4改变为主。FGF-21表达改变对肝细胞葡萄糖摄取无显著影响(P>0.05);过表达FGF-21的肝细胞内PPARγ、PPARα、羟甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)表达降低,而低密度脂蛋白受体(low density lipoprotein receptor, LDLr)表达升高;表达缺陷时则相反(均P<0.05);而对肝细胞内GLUT-1、GLUT-4、IRS-1以及葡萄糖激酶(glucokinase, GK)的mRNA表达无显著影响。而FGF-21表达改变对肝细胞内Visfatin的mRNA表达无显著影响。结论:在肝细胞中,FGF-21主要通过betaklotho和FGFR4发挥生物学作用,可能通过影响PEPCK表达调控肝细胞糖异生;并主要通过调节肝细胞HMGCR和LDLr表达从而影响胆固醇合成和清除。更多还原

【Abstract】 PARTⅠCONSTRUCTION OF FIBROBLAT GROWTH FACTOR-21 SHRNA RECOMBINATED VECTOR IN MICEObjective: To establish fibroblast growth factor-21(FGF-21) shRNA recombinated vector in mice.Methods: Two reverse repeated motifs targeting of FGF-21 gene were designed and synthesized respectively and inserted into plasmid pGenesil1.2 for generate the recombinated vectors. The effective rate of shRNA-vector (pGenesil-FGF21) was verified by Double-Fluorescence Reporter assay system.Results: Fluorescence ratio of GFP/RFP in two groups were lower than that in HK group (P<0.05), and the lowest in the pGenesil-FGF21-1 group.Conclusion: FGF-21 shRNA recombinated vector, pGenesil-FGF21, was established successfully. PARTⅡTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN 3T3-L1 ADIPOCYTESObjective: To investigate the effects of FGF-21 on glucose turnover, triglyceride metabolism and the underlying mechanisms in 3T3-L1 adipocytes.Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into the differentiated 3T3-L1 adipocytes, respectively.The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-Deoxy[3H]-D-glucose (glucose uptake rate, GUR) were evaluated by liquid scintillation counting method (LSCM).The cellular triglyceride (TG) content was measured with a colorimetric assay. The mRNA expressions of betakloth, FGFRs, GLUT-1, GLUT-4, IRS-1,PPAR-α, PPAR-γ, HSL, ATGL, ap2, adiponectin, visfatin and Leptin were detected by real-time quantitative PCR.Results: In 3T3-L1 adipocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 77.8% in mRNA expression and 75.3% in protein contents (both P<0.05). The upregulation of FGF-21 markedly increased GUR and decreased intracellular TG content (both P<0.05). The betaknotho and FGFR1 mRNA expressions were increased in FGF-21 upregulated adipocytes (both P<0.05). Meanwhile, the upregulation of GLUT-1, IRS-1, PPARγ, PPARα, ATGL, HSL, ap2 and leptin mRNA expressions were also observed (P<0.05). The reverse changes happened in FGF-21 deficient adipocyte except IRS-1.Conclusion: In 3T3-L1 adipocytes, the effect of FGF-21 expression change is mainly through betaklotho and FGFR1. FGF-21 may be related to glucose homeostasis and accommodate intracellular fat content in adipocyte.PARTⅢTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN HEPA1-6 HEPATOCYTESObjective: To investigate the roles of FGF-21 on glucose metabolism, cholesterol metabolism and the underlying mechanisms in Hepa1-6 hepatocytes.Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into Hepa1-6 hepatocytes, respectively. The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-deoxy-[3H]-D-glucose (GUR) were evaluated by liquid scintillation counting method. The mRNA expressions of betaklotho, FGFRs, GLUT-1, GLUT-4, IRS-1,PPARα, PPARγ, PEPCK, HMGR, LDLr and visfatin were detected by real-time quantitative PCR.Results: In Hepa1-6 hepatocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 86.3% in mRNA expression and 77.3% in protein contents (both P<0.05). FGF-21 has no effect on GUR. The betaknotho and FGFR4 mRNA expressions were increased in FGF-21 upregulated hepatocytes (both P<0.05). Meanwhile, FGF-21 over-expression reduced PPARγ, PPARα, HMGR and PEPCK mRNA expression and increased LDLr expression (P<0.05), but had no effect on GLUT-1, GLUT-4, IRS-1, GK and visfatin expression in Hepa1-6 hepatocytes.Conclusion: The effect of FGF-21 expression change is mainly through betaklotho and FGFR4 in hepa1-6 hepatocytes. FGF-21 maybe plays an important role in cholesterol metabolism and liver gluconeogenesis, but no glucose uptake.更多还原