信号通路与肿瘤.pptVIP

信号转导的研究方法与工具 当前31页，共47页，星期日。 一、蛋白质磷酸化状态的检测 1、免疫印迹 (phospho-protein specific antibodies) 2、免疫沉淀 (protein-specific antibody + phospho-AA antibody 3、流式细胞仪分析 4、Luminex分析 二、信号转导分子过度表达或过度激活 1、Overexpression by gene transduction 2、Constitutively activated mutants 三、基因转录活性测定 1、Electrophoretic mobility shift analysis (EMSA) ２、Reporter gene expression detection 当前32页，共47页，星期日。 四、信号转导分子的表达或活性抑制 1、Anti-sense 2、RNAi 3、Gene knock-out 4、Dominant negative mutants Ligand-binding site Phosphorylation site Docking site Protein-protein binding site DNA binding site 5、Small-molecule inhibitors：e.g., tyrosine kinase inhibitor (TKi) 6、Inhibitory oligopeptides 当前33页，共47页，星期日。 信号转导在肿瘤发生发展中的意义 当前34页，共47页，星期日。 Signaling molecules involved in cancer development/progression Receptors Growth factor receptors: EGFR Hormone receptor: ER, AR Angiogenic receptros: VEGF, PDGF, IGF Death receptors The Integrin system 当前35页，共47页，星期日。 Transducers Ras Raf Rho family PI-3K/Akt Death transducers STAT-3 …… Transcription factors c-Myc c-Jun and c-fos STAT-3 …… 当前36页，共47页，星期日。 Biological Effects of Signaling Related to Cancer Development/Progression Cell immobilization Abrogation of apoptosis Activation of cell cycle and removal of cell cycle checkpoints Angiogenesis Cell invasion Metastasis Drug resistance 当前37页，共47页，星期日。 当前38页，共47页，星期日。 Phosphorylation targets of PI-3K Akt ? Forkhead-related transcription factor 1?(FKHR-L1) ? 14-3-3 binding ? FKHR-L1 retaining in cytosol ? abrogation of gene activation by FKHR-L1 Akt ? Bad ? 14-3-3?binding ? Release of Bcl-2 and Bcl-X ? Cell survival Akt ?GSK3 ?GSK3 catalytic activity turned off ? Permitting activation of c-Myc and cyclin D PDK1 ? phosphorylation of other kinases (p70 S6-kinasse, CISK, PKC) ? Cell growth and survival 当前39页，共47页，星期日。 信号转导与肿瘤临床——诊断、预防与治疗 Expression level, mutations and antibodies of signaling molecules in cancer diagnosis EGFR: lung, HN, glioma… TGF-? receptor (type II): lung, HN, Ras Androgen receptor and dow