【作者】 周磊；

【导师】 杨在清；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 脂肪组织是机体能量代谢和平衡的主要器官，它能合成和存储甘油三脂，并在需要时分解利用脂肪酸来维持机体能量的平衡。近年来的研究表面，脂肪组织还能产生和分泌各种信号分子，调节整个机体的代谢活动。这些具有生物学活性的信号分子称脂肪细胞因子(adipokines)，包括TNFα、Leptin、Adiponectin(也被称为AdipoQ或Acrp30)及Resistin(也称ADSF或FIZZ3)等。这些因子在脂肪组织中表达量很高，而且大部分脂肪细胞因子在脂类代谢、进食、能量平衡、炎症以及胰岛素抗性的调控中起着重要作用。抗素是脂肪细胞因子家族的一个重要成员，从发现之日起就受到人们的重视。已有的研究表明，抗素在炎症、葡萄糖耐受、胰岛素抵抗等方面具有重要作用。但抗素在正常生理状态下的功能，作用的靶组织，是否存在受体以及信号调控等问题都有待回答。本研究以小鼠和人的抗素作为研究对象，采用半定量RT-PCR、Western Blotting和细胞转染等技术，对抗素的功能、转录和翻译水平的表达调控等进行了研究，获得如下结果：1．通过同源分析比较，以大鼠抗素剪切体为参照，运用RT-PCR方法，获得了小鼠抗素的剪切体；以小鼠的心、肝、脾、和肺等九个组织样品作为研究材料，通过半定量RT-PCR分析获得了剪切体的组织表达谱；利用生物信息学方法对小鼠、大鼠和人的剪切体进行了比较分析，确定小鼠的剪切体既不具有分泌能力，也不能编码翻译蛋白质。2．采用RT-PCR方法，克隆了SREBP、CREB、C／EBPα、抗素的编码序列和抗素基因的启动子序列。将其分别构建真核表达载体，通过转染和共转染研究了它们对抗素转录表达的影响。结果表明，C／EBPα可增强抗素的表达，而SREBP和CREB对抗素的表达无影响。3．以巨噬细胞为模型，研究了胰岛素、罗格列酮、脂多糖、地噻米松和克伦特罗等激素类效应物对抗素基因表达的影响。结果表明，低浓度的胰岛素抑制抗素和TNFα的表达，增强PPARγ表达，而高浓度的胰岛素促进抗素和TNFα的表达，抑制PPARγ表达；罗格列酮抑制抗素的表达；脂多糖、地噻米松和克伦特罗都增强抗素和TNFα的表达。这些结果与抗素在脂肪细胞中的研究结果一致，说明在巨噬细胞和脂肪细胞中抗素基因表达具有相同的调节方式。4．通过基因转染，抗素基因在肝细胞中过量表达，可显著的降低细胞对葡萄糖的利用，增加控制肝糖生成的G6Pase的表达量，降低胰岛素敏感性，但控制葡萄糖进入肝细胞的GLUT2表达量则无变化，这表明，抗素超表达引起的离体肝细胞胰岛素抗性可以不涉及GLUT2表达量变化。5．Western Blotting分析表明，用脂肪细胞条件培养基处理肝细胞，可显著的降低肝细胞的胰岛素敏感性；而用不同浓度的抗素重组蛋白处理肝细胞，也使肝细胞中胰岛素信号途径的敏感性下降，而且具有浓度依赖性；说明单一的重组抗素可剂量依赖性的再现整体脂肪细胞分泌因子引起的肝细胞胰岛素抗性。因此，单一的重组抗素可以作为肝细胞胰岛素抗性模型的诱导因子。6．以不同浓度抗素重组蛋白对肝细胞进行时间梯度处理，用Western Blotting方法分析抗素对肝细胞信号物质表达和活性的影响。结果表明，抗素能快速激活Erk，增强p65的表达水平，但降低AMPK和ACC的磷酸化水平。这些因子的变化可能与抗素降低肝细胞葡萄糖耐受和增强胰岛素抵抗的应答有关。更多还原

【Abstract】 Adipose tissue (AT) is a major metabolic organ. It can synthesize and storetriglyceride, and release it when organ needs to maintain energy balance. AT can secrete anumber of adipokines, such as TNFα, leptin, 1L-6, adiponectin and resistin etc. Theseadipokines express greatly in AT and are involve in regulating insulin action and glucosemetabolism linking to obesity and diabetes. The insulin resistance is namely theattenuation of the insulin physiological functions. Current studies indicated that manyadipokines were important in lipid metabolism, energy balance and insulin resistance.Resistin was a newly found adipokine. Researchers found it when use rosiglitazone totreat 3T3-L1 cells. Current studies indicated that resistin played key roles in inflammation,glucose tolerance and insulin resistance. However, resistin function in normal status,resistin’s target, resistin’s acceptor and signaling regulation need been clarified further.In this study, by choosing mouse and human resistin as subjects and usingsemi-quantitative RT-PCR, western blotting and transfection, we studied thetranscriptional regulation, expressional regulation and function of resistin and got thefollowing results:1. By the comparative analysis and semi-quantitative RT-PCR, according rat resistinsplicing mechanism, mouse resistin splicing form was cloned and the expressionpattern of resistin splicing form in nine tissue samples was determined. By analyzingmouse resistin splicing form sequence and comparing with rat and human resistinsplicing form, mouse resistin splicing form was found neither secretes, nor codesprotein.2. By RT-PCR, CDS of SREBP, CREB, C/EBPαand resistin were cloned; by PCR, thepromoter of resistin was cloned. Subsequently, their expression vectors wereconstructed, respectively. By transfection or co-transfection, their effects ontranscription of resistin were investigated. The results indicated that C/EBPαcouldenhance resistin transcription, but SREBP and CREB could not.3. By using insulin, rosiglitazone, LPS, dexamethasone and clenbuteroi, the effects on resisitn expression in macrophages were explored. Our data showed that lowerconcentration insulin inhibited expression of resistin and TNFα, stimulated PPARγexpression; high concentration insulin increased expression of resistin and TNFα,suppressed PPARγexpression. And rosiglitazone decreased resistin expression. ButLPS, dexamethasone and clenbuterol increased expression of resistin and TNFα.These data were the same as that obtained from adipocytes. It suggests resistin has thesame regulated mechanism between adipocytes and macrophages.4. By transfection, resistin was overexpressed in hepatic cells. Resistin overexpressionrepressed glucose uptake dramatically. By analyzing the expression of relative genes,the data indicated G6Pase expression was enhanced which controls hepatic glucoseproduction; however GLUT2 expression had no change which transfers glucose fromextracellular to intracellular. And the results demonstrated resistin overexpression alsoimpaired insulin sensitivity in hepatic cells.5. By western blotting, the effect of adipocyte-conditioned medium (CM) on hepaticinsulin resistance was monitored. The data showed CM induced a marked hepaticinsulin resistance. Subsequently, to investigate the role of recombinant resistin in thisprocess, its effect on insulin signal were measured. The results demonstratedrecombinant resistin suppressed insulin action and induced insulin resistance inhepatocyte. And its effect was dose-dependent. Therefore, recombinant resistin canuse to build insulin resistance model in hepatocyte.6. With different time and different dose treatment, by western blotting, the effects ofresistin on other signal pathways in hepatocytes were determined. It showed resistinactivated Erk, enhanced p65 expression and impaired phosphorylation of AMPK andACC. These might be the reason to explain why resistin could impair glucosetolerance and insulin sensitivity.更多还原