如何探讨中药有效成分对基因调控的影响.pptVIP

如何探討中藥有效成分對基因調控的影響 Electrophoretic Mobility Shift Assay (EMSA) 研究DNA結合蛋白與其相關結合序列相互作用的方法 Protein / DNA (transcription factor) / (promoter region) EMSA Nuclear protein DNA probe Native gel (non-denaturing agarose gel) Autoradiography Promoter activity Promoter Regulation of atherosclerosis-related genes by herbal medicines : Wog inhibits MCP-1 and TF gene expressions Protocol for gene regulation ? mRNA ― Northern Blot ? mRNA stability ― Northern Blot ? protein ― ELISA or Western Blot ? promoter activity ― Luciferase activity ? activation of transcription factor ― EMSA ? signal transduction ― Western Blot Atherosclerosis a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries ? classic concept : as a disorder of lipid metabolism and deposition ? current view : as a chronic inflammatory disease Three stages in the life history of an atheroma ? ?? Initiation : permeability/ adhesion/ migration ??? ? Progression : fatty-streak formation/ advanced lesion ??? ? Complication : plaques rupture 中醫的觀點： 《外科正宗》--- 厚味膏梁, 薰蒸臟腑, 丹石補藥 消灼腎水; 房勞過度, 氣竭津傷, 致使經絡阻塞, 氣血凝滯而發病。 屬中醫“脈痺” 和“脫疽”的範疇 血瘀證 * * Stimulation Inflammatory protein Signal transduction Transcription factor Protein kinase phosphorylation Protein-DNA binding Protein mRNA AP-1 NF-?B AP-1 NF-?B Egr-1 MCP-1 TF (Western Blot) *(EMSA) (Northern Blot) (ELISA) (ex. ERK, P38, JNK) (ex. AP-1,NF-?B,Egr-1) Receptor Gene Regulation Membrane Nuclear ex. TNF, IL-1, MCP-1, ICAM-1 ex. cytokine, LPS, stress CM98/03/07/94 Reporter gene Luciferase 　　　　　　　　　Luciferase Lucoferin + ATP Light Gene expression in the development of atherosclerosis Initiation Progression Complication MCP-1 MMP TF VC