【作者】 张静；

【导师】 刘国良；

【作者基本信息】 中国医科大学 ， 内科学， 2004， 博士

【摘要】 目的 胰岛素抵抗是指一定量的胰岛素与其特异性受体结合后生物效应低于正常，表现为外周组织尤其是肌肉、脂肪组织对葡萄糖摄取减少及抑制肝葡萄糖输出的作用减弱，临床上表达出糖代谢紊乱、肥胖、血脂异常等多代谢失调。胰岛素抵抗是2型糖尿病发病的基础。肥胖是2型糖尿病的重要危险因素，肥胖和2型糖尿病患者中普遍存在着胰岛素抵抗。近年发现肥胖症和2型糖尿病患者体内游离脂肪酸（free fatty acid，FFA）的升高在胰岛素抵抗的发生过程中起了重要作用。 胰岛素的主要生理作用是调节靶组织对葡萄糖的利用，维持体内糖代谢的平衡，而其生物学效应的实现依赖于胰岛素信号传导通路。胰岛素的代谢效应主要与胰岛素信号传导的磷脂酰肌醇-3激酶（phosphatidylinosi-tol-3 kinase，PI-3K）途径有关，胰岛素与靶细胞表面的胰岛素受体（IR）α亚单位结合，使IR的β亚单位自身磷酸化和激活其内在的酪氨酸激酶，导致胰岛素受体底物（IRS）的酪氨酸残基磷酸化，磷酸化的IRS与PI-3K的p85调节亚单位结合，解除p85调节亚单位对PI-3K的p110催化亚单位的抑制而激活PI-3K，进而使蛋白激酶B（protein kinase B，PKB）磷酸化而激活。PKB为PI-3K通路中的关键分子，它能够作用于糖原合酶激酶-3（GSK-3）、葡萄糖转运子（GLU7）、p⁷⁰S6激酶(p⁷⁰S6K)等多种底物，可介导产生多种生物学效应，如糖原合成、蛋白合成、葡萄糖转运、抑制细胞凋亡等。在实现胰岛素生物学效应过程，胰岛素信号转导的多个环节受到信号蛋白的反馈调控和制约，处于下游的多种丝（苏）氨酸激酶，对胰岛素信号转导起负性反馈调节作用；同时，下游的丝（苏）氨酸激酶，主要是PKB，能够维持IRS的活化构象，可对信号系统起正性反馈作用。信号转导系统的某些环节如受到阻碍，级联反应失去平衡，则出现不同程度的生物学效应降低，即胰岛素抵抗。 葡萄糖进入细胞不是单纯扩散的过程，而是由蛋白质载体-GLUT的介导从而易化葡萄糖穿过细胞膜进入细胞内，这一过程是葡萄糖代谢中的限速步骤。葡萄糖转运子4（GLUT4）是现已发现的十余种GLUT中的重要一员，仅存在于胰岛素敏感的骨骼肌、心肌和脂肪细胞中，胰岛素刺激后GLUT4由细胞内的贮存囊泡中转位至细胞外膜，且活性增加，与葡萄糖结合并发生结构改变，将葡萄糖转运至细胞内后恢复原来结构。由此胰岛素敏感组织可迅速对循环胰岛素水平作出反应，保持血糖平衡。因此GLUT4的表达在蛋白水平和基因水平都得到严密的调控，而能够影响其表达及转位的因素则可能导致葡萄糖跨膜转运的异常，从而导致胰岛素抵抗。 由于F队升高对不同的靶组织产生的影响不同，因而离体实验是研究FFA对不同组织影响的作用机制的很好的工具。脂肪组织是胰岛素的重要靶组织，近年发现它不但能储存能量，还是内分泌器官，能分泌产生瘦素（玩ptin）、抵抗素（Resistin）、核转录因子ppA彻、肿瘤坏死因子一。（TNF-a）等多种激素和细胞因子，与肥胖和2型糖尿病密切联系，在胰岛素抵抗的发病机制的研究中有重要的地位。 胰岛素抵抗的病理机制的研究需要可靠的体外模型。3竹一Ll细胞株是国外应用较广泛的重要细胞模型，在一定的体外培养条件下能够由成纤维细胞分化为成熟脂肪细胞，对胰岛素的刺激敏感，主要用于胰岛素刺激的葡萄糖摄取和代谢的信号传导机制及降糖药物作用机制的研究;而国内有关3竹一L1细胞的研究报道还为数不多。 骨髓间充质干细胞（mesenehymal stem eells，MSC）来自于骨髓基质，又称为“骨髓基质细胞”（bone marrow stromal eells，BMSC），对骨髓造血干细胞起支持诱导作用。在不同的诱导条件下，MSC具有向中胚层组织细胞分化的能力，如向成骨细胞、成软骨细胞、脂肪细胞、成纤维细胞等分化的能力。 本研究利用3竹一L1细胞体外诱导分化为脂肪细胞;同时用密度梯度离心和贴壁培养方法对大鼠MSC进行分离培养和表面抗原鉴定，建立体外定向诱导MSC分化为脂肪细胞的方法;并对这两种通过体外定向诱导分化来得到大量成熟脂肪细胞的方法加以比较，探索以脂肪细胞为模型研究其在内分泌系统的作用的新途径。本研究还选择了有代表性的FFA一软脂酸（palmitie aeid，PA）和油酸（oleie aeid，OA），观察不同种类和浓度的FFA对3竹一Ll脂肪细胞葡萄糖摄取及胰岛素敏感性的影响，建立FFA诱导的胰岛素抵抗的细胞模型;并进一步观察FFA对胰岛素信号转导通路中的关键蛋白PI一3K和PKB的蛋白表达和PKB磷酸化水平（p一PKB）的影响、FFA对GLUT4基因表达、蛋白表达及转位的影响。方法 1 .3竹一U细胞的培养及诱导分化 小鼠3竹一Ll细胞在含10%小牛血清、looU/耐青霉素和100林扩而链霉素的DMEM培养液中，在37℃、5%CO:、饱和湿度条件下常规培养。细胞汇合达90%以上后，开始进行诱导分化。无血清DMEM培养液孵育2小时后，加人含1林moFL的地塞米松、0 .5 mmoFL的IBMX、1林扩祖的胰岛素、10%FBS的DMEM（诱导培养液l）诱导48h，含l林扩耐的胰岛素、10%FBS的 DMEM（诱导培养液2）处理48h，之后改为含10%FBS的DMEM（维持培养液）更多还原

【Abstract】 ObjectiveInsulin resistance is a major characteristic of Type 2 diabetes, and is also commonly associated with obesity, hypertension and cardiovascular disease. It can be defined as a failure of target tissue to increase whole body glucose disposal in response to insulin. The elevated levels of plasma free fatty acids ( FFA) noted in patients with Type 2 diabetes and obesity may lead to insulin resistance.The latest developments in insulin signaling cascade show that insulin stimulated - glucose metabolism is mainly involved in phosphatidylinositol - 3 ki-nase (PI-3K) pathway. A fundamental event , following the binding of insulin to insulin receptor(IR) , is the activation of the intrinsic tyrosine kinase, leading to autophosphorylation. This in turn enables a number of docking proteins, including insulin receptor substrate - 1 (IRS - 1) , - 2, to be recruited. Activation of PI-3K through binding of its p85 adaptor subunit to IRS -1 raises the levels of several second messengers and hence activated protein kinase B (PKB). PKB has been implicated in the translocation of glucose transporter 4 (GLUT4) to the plasma membrane, which underlies insulin - stimulated glucose uptake. Therefore, any factor that alters the insulin signalling pathway and the translocation of GLUT4 may lead to the attenuation of insulin signal resulting in insulin resistance.Elevated FFA levels may exert multiple negative effects, depending on the differentenl target tissue. Therefore, the ungerlying intracellular signaling mechanisms that are affected by elevated FFA levels are best deciphered in vitro.3T3-L1 cells represent an useful model for studies of insulin action, particularly glucose metabolism, since these cells can differentiate from fibroblasts to adipocytes that are sensitive to insulin under appropriate culture conditions.Mesenchymal stem cells (MSC) is separated from bone marrow and contributes to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, adipose, etc.In this study, two kinds of adipocytes differentiated from 3T3-L1 cells and rat MSCs were cultured, stained and compared. After 3T3-L1 adipocytes were treated by palmitic acid (PA) or oleic acid(OA) , glucose uptake were measured to examine the FFA - induced cell model of insulin resistance. The protein expression of PI-3K, PKB, phosphated - PKB ( p - PKB) and GLUT4 were further detected; as well as the mRNA expression and the translocation of GLUT4.Method1. 3T3-L1 cells were differentiated into adipocytes in DMEM supplemented with dexmethason, IBMX, insulin andlO%FBS.2. A density gradient was used in the isolation and purification procedure of the rat MSCs. Detection of surface antigen of cultured MSCs were conveyed with immunocytochemistry method. MSCs were differentiated into adipocytes in DMEM supplemented with dexmethason, IBMX, insulin andlO%FBS.3. Oil Red 0 stain was performed to examine the differentiated adipocytes.4. 3T3-L1 adipocytes were incubated with PA or OA of different concentration for different duration.5. 3H - DG glucose uptake was measured by scintillation counting method, nonspecific 3H - DG uptake were measured in the presence of cytochalasin B.6. Total cell protein was extracted from the FFA - treated 313-LI adipocytes and controls.7. Western blot analysis was employed to detect the protein expression of PI-3K p85a, PKB -a, p -PKB and GLUT4.8. Total RNA was extracted from the FFA - treated 3T3-L1 adipocytes and controls. Semi - quantitative RT-PCR was used to detect the mRNA expression of GLUT4 with the house - keeping gene p - actin as intrinsic control.9. Indirect immunofluorescence was processed to observe the translocation of GLUT4 of the FFA - treated 3T3-L1 adipocytes and controls.10. Immunocytochemistry method was processed to quantitatively analysis the translation of GLUT4 of the FFA - treated 313-LI adipocytes and controls.Result1. 3T3-L1 cells were differentiated into adipocytes and approved by Oil Red 0 stain at a rate rang更多还原