Trx真核表达载体的构建及ROS调控INS-1细胞胰岛素分泌机制的初步研究-生物化学与分子生物学专业论文.docxVIP

II II Abstract The regulation of insulin secretion was very important to diabetes therapy. ROS as signal molecules participate in the regulation of insulin secretion, but the underlying mechanism is unclear. PTEN, a kind of tyrosine phosphates of β cell, can negatively regulate PI3K/Akt signal pathway, which affect insulin secretion through down-regulation of Akt phosphorylation. Cys124 as an active site of PTEN, can be oxidated by ROS and then induces the inactivation of PTEN. However, Trx can recover the oxidation of PTEN. In this study, Trx Eukaryotic Expression Vector was successfully constructed and evaluated by gene sequencing analysis, restriction enzyme analysis and Western blot analysis. In addition, the mechanisms of insulin secretion regulated by H2O2 were primarily studied. The intracellular ROS content, insulin secretion and PTEN protein expression and oxidation in INS-1 cells stimulated by various concentrations of H2O2 were evaluated using DCFH-DA fluorescent probe, RIA and Western blot analysis, respectively. The results showed that 5 - 400 μM H2O2 increased intracellular ROS level in a concentration-dependent manner. Low level of H2O2（≤100 μM）increased insulin secretion. However, high level of H2O2 ( 100 μM ) inhibited insulin secretion. Additionally, 5 - 400 μM H2O2 increased intracellular PTEN protein expression and oxidation in a concentration-dependent manner. 1 mM H2O2 down-regulated PTEN protein expression. These results provided solid base for further research on the relationship between ROS and insulin secretion. Keywords: Trx insulin secretion ROS PTEN INS-1 PAGE PAGE IV 目 录 摘 要I ABSTRACT II 1 绪论 1.1 胰岛素分泌与糖尿病 (1) 1.2 活性氧与胰岛素分泌的关系 (2) 1.3 蛋白质磷酸化、PTP 与胰岛素分泌的关系 (3) 1.4 抗氧化酶与胰岛素分泌的关系 (6) 1.5 研究目的及内容 (7) 2 PCDNA3.0-TRX 真核表达质粒的构建与表达检测 2.1 引言(9) 2.2 实验材料(9) 2.3 TRX 真核表达载体构建 (11) 2.4 实验结果(25) 2.5 本章小结(27) 3 活性氧调控 INS-1 细胞胰岛素分泌的机制初探 3.1 引言(28) 3.2 实验材料(28) 3.3 实验方法(31) 3.4 实验结果(33) 3.5 本章小结(36) 4 全文总结及展望 4.1 全文总结(38) 4.2 展望(