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背景:表皮生长因子和胰岛素都主要通过PI3K通路和MAPK通路进行信号转导,但各自的生理功能不同。目的:从细胞整体水平对比分析小鼠肝细胞内胰岛素与表皮生长因子信号转导磷蛋白质组的动力学行为差别,以此找出二者关键性的信号蛋白。设计、时间及地点:随机分组设计的对比观察实验,于2005-07/2006-04在泸州医学院分子生物学实验室完成。材料:实验用肝细胞来源于昆明种封闭群乳小鼠。方法:选取细胞长势相当的肝细胞,采用放射性同位素(32P)对其进行标记,标记后按随机数字表法分为3组,空白对照组、表皮生长因子刺激组(给予10μg/L表皮生长因子)及胰岛素刺激组(给予100nmol/L胰岛素)。主要观察指标:表皮生长因子、胰岛素分别作用0,5,20,60,120min,采用双向电泳技术分析比较表皮生长因子与胰岛素分别介导的磷酸化蛋白质组的动力学行为,即磷酸化水平。结果:参与两者信号转导的磷蛋白质种类没有多大差异,大多数信号磷蛋白磷酸化水平的动力学行为也表现为一致性,但有4个蛋白点其磷酸化水平随时间变化趋势明显不同。结论:胰岛素与表皮生长因子在同一细胞内参与信号转导的个别信号蛋白其磷酸化水平的动力学行为差别较大,估计这些蛋白就是导致表皮生长因子与胰岛素最终生物学活性的关键蛋白。
Background: Both epidermal growth factor and insulin signal transduction through the PI3K pathway and MAPK pathway, but their physiological functions are different. OBJECTIVE: To compare the dynamic behavior of insulin and epidermal growth factor signal transduction phosphoproteome in mouse hepatocytes from the overall cellular level to find out the key signaling proteins. DESIGN, TIME AND SETTING: The comparative observation experiment with randomized block design was performed at the Laboratory of Molecular Biology, Luzhou Medical College from July 2005 to April 2006. MATERIALS: Experimental hepatocytes were derived from Kunming hermaphroditic mice. Methods: Hepatocytes with similar cell growth were selected and labeled with radioisotope (32P). The cells were divided into 3 groups according to random number table. The blank control group, epidermal growth factor stimulation group (10μg / L epidermal growth factor ) And insulin stimulation group (given 100nmol / L insulin). MAIN OUTCOME MEASURES Epidermal growth factor and insulin were respectively treated for 0, 5, 20, 60 and 120 min. The kinetic behavior of phosphoproteomes mediated by epidermal growth factor and insulin was analyzed by two-dimensional electrophoresis, that is, phosphorylation level. Results: There was no significant difference in the types of phosphoproteins involved in signal transduction between the two groups, and most of the phosphorylated phosphorylation levels showed a consistent kinetic behavior. However, the phosphorylation levels of four protein spots changed significantly with time . CONCLUSION: The dynamics of phosphorylation of individual signaling proteins involved in signal transduction between insulin and epidermal growth factor are quite different. It is estimated that these proteins are the key proteins leading to the final biological activity of epidermal growth factor and insulin.