如何探讨中药有效成分对基因调控的影响.ppt

如何探討中藥有效成分對基因調控的影響
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
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
Reporter gene
Luciferase
Luciferase Lucoferin + ATP Light
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