【作者】 郭晓农；

【导师】 贾正平； 张汝学；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 目的:本课题以中药地黄的提取物地黄寡糖为研究对象,围绕2型糖尿病的主要病理生理学机理——胰岛素抵抗,采用体外细胞模型,运用现代药理学和分子生物学的研究方法,探讨研究地黄寡糖改善胰岛素抵抗的作用及分子机理。 方法:培养3T3-L1前脂肪细胞,用四甲基偶氮唑盐（MTT）方法检测3T3-L1前脂肪细胞及脂肪细胞的增殖、分化情况:同时采用地塞米松诱导3T3-LⅠ脂肪细胞建立胰岛素抵抗模型;检测地黄寡糖对细胞培养基中葡萄糖浓度的影响;运用RT-PCR技术,检测地黄寡糖对脂肪细胞内一些关键基因表达的影响,包括转录调控因子,调控细胞分化、代谢的过氧化物酶体增殖物激活受体-γ（PPAR-γ）;调节体内脂代谢的重要细胞因子脂联素（adiponectin）;脂肪细胞分化标志基因脂肪酸结合蛋白（ap2）;调节体内葡萄糖代谢的重要细胞因子葡萄糖转运蛋白4（GLUT4）及脂肪细胞分泌的能诱导胰岛素抵抗产生的重要基因抵抗素（Resistin）。 结果:在DMEM高糖培养基中,地黄寡糖(0.1～30μg·ml^-1)可促进3T3-L1前脂肪细胞增殖,抑制3T3一L1脂肪细胞增殖,作用呈明显量效关系;使3T3-L1前脂肪细胞及3T3-L1脂肪细胞葡萄糖消耗量增加,呈明显量效关系;抑制3T3-L1前脂肪细胞的分化。地黄寡糖(0.1～30μg·ml^-1)能明显增加胰岛素抵抗3T3-L1脂肪细胞对葡萄糖的摄取,增强对胰岛素的敏感性,改善地塞米松诱导的3T3-L1脂肪细胞的胰岛素抵抗。地黄寡糖(0.1～30μg·ml^-1)能够促进胰岛素抵抗3T3-L1脂肪细胞中PPAR-γ及其下游基因adiponectin、ap2、GLUT4 mRNA的表达,明显降低Resistin基因mRNA的表达。 结论:地黄寡糖可以促进前脂肪细胞的增殖,抑制前脂肪细胞的分化,抑制脂肪细胞的增殖,地黄寡糖对地塞米松诱导的3T3-L1脂肪细胞胰岛素抵抗具有明显的改善作用,其机制可能与激活PPAR-γ,并调控PPAR-γ下游靶基因mRNA的表达有关。该项研究初步阐明了地黄寡糖改善胰岛素抵抗的作用及其分子机理,为地黄寡糖防治2型糖尿病胰岛素抵抗提供了有益的数据。更多还原

【Abstract】 Objective:Rehmannia glutinosa Libosch. is a traditional Chinese medicinal drug and has been used for treatment of type 2 diabetes in China for thousands of years. This thesis is to study the ameliorative effect of Rehmannia glutinosa oligosaccharides （ROS） on insulin resistance, which is one of the key steps of pathophysiological mechanism in the natural history of type 2 diabetes, in cellular models induced by dexamethasone by means of pharmacological and molecular biological methods.Methods:3T3-L1 preadipocytes were cultured and the differentiation and proliferation of adipocytes were detected by MTT method. Insulin resistant 3T3-L1 adipocytes cell model was induced by dexamethasone and the change of glucose concentration in cell culture was determined after ROS treatment. The mRNA expressions of peroxisome proliferator-activated receptor gamma（ PPAR-γ）,adiponectin, ap2 and GLUT4 relating to the differentiation of adipocytes and insulin seneitivity as well as Resistin that can induce insulin resistance were detected by reverse transcription PCR（RT-PCR）.Results:In the high glucose DMEM culture media, by means of MTT method, the absorbance at 570 nm of 3T3-L1 preadipocytes was increased and that of 3T3-L1 adipocytes was decreased. ROS(0.1³0 μg·ml^-1 )significantly increased glucose consumption in 3T3-L1 preadipocytes and adipocytes culture with a concentration-dependent effect. ROS enhanced the sensitivity of 3T3-L1 adipocytes to insulin and improved the insulin resistence of 3T3-L1 adipocytes induced by dexamethasone significantly. ROS(10 μg·ml^-1) can up-regulate mRNA expression of PPAR-γ, adiponectin, ap2 and GLUT4, inhibit that of Resistin.Conclusion:ROS can promote the proliferation of 3T3-L1 preadipocytes, inhibit the proliferation of 3T3-L1 adipocytes and significantly improve insulin resistance induced by dexamethasone. The roles of improving insulin resistence may related to the activation of PPAR-γ and regulation of related mRNA gene expressions of of adiponectin, ap2 and GLUT4, as well as inhibition of Resistin gene expression that can induce insulin resistence. The study primalarily illustrates the ameliorative effects of ROS on insulin resistance in vitro and its mechanism, demonstrates its values of potential clinical application for prevention and treatment of type 2 diabetes mellitus in the future.更多还原