【作者】 李仁哲；

【导师】 李伶；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2008， 硕士

【摘要】 目的:构建大鼠内脏脂肪素(visfatin)基因pcDNA3.1(+)真核表达载体并转入大鼠,观察visfatin基因过表达对正常饮食及高脂饮食大鼠胰岛素敏感性,FGF-21和糖脂代谢的影响。方法:1、构建pcDNA3.1(+)– visfatin真核表达质粒。2、将pcDNA3.1(+)-visfatin质粒转入正常饮食(NT组)及高脂饮食(HT组)大鼠,并以正常饮食pcDNA3.1(+)空质粒(NC组)为对照,采用正糖-高胰岛素钳夹技术评价大鼠胰岛素敏感性改变,同时测定血糖、血脂、血浆visfatin和FGF-21的变化。3、用dot-blot方法测定肝脏、脂肪、肌肉组织IRS1的磷酸化水平。4、用RT-RCR和Westernblot方法测定肝脏和脂肪组织脂代谢相关基因及蛋白水平的变化。结果:1、成功构建了重组质粒pcDNA3.1(+)- visfatin。2、质粒转染后3天,NT组和HT组大鼠血浆visfatin水平均明显升高(P<0.01),NT组血浆基础胰岛素水平明显降低(P<0.05),葡萄糖输注率较转染前显著升高(P<0.01)。肝脏、脂肪、肌肉组织IRS1磷酸化的水平明显增高(P<0.05),NT组和HT组大鼠肝脏PPARγmRNA的表达均明显升高(分别为P<0.05和P<0.01),NT组大鼠脂肪组织PPARγmRNA亦显著升高(P<0.01),而HT组则仅有升高趋势(P>0.05);3、与质粒转染前相比,各处理组大鼠血浆总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)水平明显降低(P<0.05)。SREBP2 mRNA表达均明显高于对照组(P<0.05), NT组HMG CoA还原酶mRNA水平明显高于NC组和HT组(P<0.05),NC组与HT组无明显差异;4、NT组大鼠脂肪组织ATGL mRNA及蛋白水平均明显高于NC组和HT组(P<0.05),且HT组高于NC组,但无统计学意义。5、与质粒转染前比较,NT组大鼠血浆FGF-21水平明显下降(P<0.05),而HT组大鼠血浆FGF-21水平明显升高(P<0.05);肝脏FGF-21 mRNA及蛋白水平变化与血浆FGF-21变化趋势一致。结论:1、成功构建了重组质粒pcDNA3.1(+)-visfatin。2、重组质粒使血浆visfatin水平明显升高,增强了大鼠胰岛素敏感性,血浆总胆固醇水平明显下降,并提高了脂肪组织ATGL mRNA及蛋白水平,同时对FGF-21的表达产生极大的影响。更多还原

【Abstract】 Objective: To construct the eukaryotic expression vector of visfatin (pcDNA3.1 (+)-visfatin) and transfect it into normal and high fat-diet rats; To investigate the effect of visfatin gene overexpression on insulin sensitivity, glucose and lipid metabolism and cytokine in these rats.Methods: 1. The recombinant vector (pcDNA3.1 (+)-visfatin) was constructed and transfected into normal and high-fat diet rats. 2. The euglycemic-hyperinsulinemic clamp experiments were performed for evaluation the change of insulin sensitivity. The effect of visfatin was evaluated on plasma glucose, lipid level, and FGF-21. 3. The phosphorylation state of IRS1 in liver, skeletal muscle and adipose tissues were determined by dot-blot. 4. The expression of lipid metabolism related mRNA and protein, such as HMG CoA reductase, SREBP2, PEPCK, SCD1 in liver and ATGL, HSL in adipose tissue, was analyzed by RT-PCR and western blot.Results: 1. Recombinant pcDNA3.1 (+)-visfatin was successfully constructed. 2. Plasma visfatin levels and glucose infusion rates(GIR) were significantly increased of NT and HT rats after 3 days of pcDNA3.1 (+)-visfatin transfection(P<0.01). Plasma fasting insulin were decreased in NT rats(P<0.05). The pIRS1 levels in hepatic, adipose and muscle tissue were significantly increased of NT and HT rats than that of NC rats(P<0.05). Hepatic PPARγmRNA levels of NT and HT rats were significantly higher than that of NC rats(P<0.05 and P<0.01). adipic PPARγmRNA levels of NT rats were significantly increased (P<0.05),and that of HT rats were higher than NC rats but no statistical difference . 3. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) were significantly decreased of NT and HT rats after 3 days of pcDNA3.1 (+)-visfatin transfection(P<0.05). Hepatic SREBP2 mRNA levels of NT and HT rats were significantly higher than that of NC rats(P<0.05). HMG CoA reductase of NT rats were significantly higher than that of NC and HT rats(P<0.05), and there is no statistical difference between NC and HT rats. 4. Adipic ATGL mRNA levels of NT rats were significantly higher than that of NC and HT rats(P<0.05), and that of HT rats were higher than NC rats but no statistical difference . 5.After 3 days of pcDNA3.1 (+)-visfatin transfection plasma FGF-21 were decreased in NT rats,however, FGF-21 were increased in HT rats(P<0.05). The diversity of hepatic FGF-21 mRNA and protein levels was similar to that of plasma FGF-21.Conclusion: 1.The eukaryotic expression vector of pcDNA3.1 (+)-visfatin was successfully constructed. 2. The transfection of pcDNA3.1 (+)-visfatin increase plasma visfatin levels, improve insulin sensitivity and decrease plasma total cholesterol. Visfatin overexpression is effective in increasing the expression of adipic ATGL mRNA and protein levels, and influencing expression of FGF-21.更多还原