CAJ | 학술논문

背景:表皮生长因子和胰岛素都主要通过P13K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10 μg/L表皮生长因子)及胰岛素刺激组(给予100 nmol/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牛长因子与胰岛素最终生物学活性的关键蛋白.
배경:표피생장인자화이도소도주요통과P13K통로화MAPK통로진행신호전도,단각자적생리공능불동.목적:종세포정체수평대비분석소서간세포내이도소여표피생장인자신호전도린단백질조적동역학행위차별,이차조출이자관건성적신호단백.설계、시간급지점:수궤분조설계적대비관찰실험,우2005-07/2006-04재로주의학원분자생물학실험실완성.재료:실험용간세포래원우곤명충봉폐군유소서.방법:선취세포장세상당적간세포,채용방사성동위소(32P)대기진행표기,표기후안수궤수자표법분위3조,공백대조조、표피생장인자자격조(급여10 μg/L표피생장인자)급이도소자격조(급여100 nmol/L이도소).주요관찰지표:표피생장인자、이도소분별작용0,5,20,60,120min,채용쌍향전영기술분석비교표피생장인자여이도소분별개도적린산화단백질조적동역학행위,즉린산화수평.결과:삼여량자신호전도적린단백질충류몰유다대차이,대다수신호린단백린산화수평적동역학행위야표현위일치성,단유4개단백점기린산화수평수시간변화추세명현불동.결론:이도소여표피생장인자재동일세포내삼여신호전도적개별신호단백기린산화수평적동역학행위차별교대,고계저사단백취시도치표피우장인자여이도소최종생물학활성적관건단백.
BACKGROUND: Both epidermal growth factor (EGF) and insulin transfer their signals into cells by two primary signal transduction pathways,including phosphatidylinositol 3-kinase (PI3K) pathway and mitogen activated protein kinases (MAPK) pathway.But they have different physiological functions.OBJECTIVE: To comparatively assay the dynamic behaviors of phosphoproteomes between EGF and insulin signal transductions in mouse hepatocytes and find key signal proteins.DESIGN,TIME AND SETTING: Randomized grouping controlled observation experiment was performed in the laboratory of Molecular Biology,Luzhou Medical College between July 2005 and April 2006.MATERIALS: Hepatocytes were from Kunming mice of closed population.METHODS: The primarily cultured mouse hepatocytes were labeled with 32p isotope and then randomly divided into three groups: control,EGF-stimulated (received 10 μg/L EGF),and insulin-stimulated (received 100 nmol/L insulin) groups.MAIN OUTCOME MEASURES: After mouse hepatocytes were treated with EGF and insulin for 0,5,20,60 and 120 minutes,the dynamic behaviors of phosphoproteomes(I.e,phosphorylated level) between EGF and insulin signal transductions were comparatively analyzed by two-dimensional electrophoresis method.RESULTS: The categories of all phosphorylated proteins between EGF and insulin-stimulated phosphoproteomes had no apparent difference.The dynamic behaviors of phosphoproteomes of most proteins during EGF signal transduction are parallel with those during insulin stimulation,except the dynamic behaviors of 4 proteins are different significantly.CONCLUSION: Aforementioned 4 phosphorylated proteins were most probably the key members that could distinguish between two signal transduction pathways ornetworks,and determined their major physiological functions respectively.