title: An in vitro analysis system using a fluorescence protein reporter for evaluating anti-inflammatory effects in macrophages.
abstract: Monitoring of inflammation in adipose tissues, which causes insulin resistance, is valuable in evaluating insulin resistance. We developed an in vitro analysis system using a fluorescence protein (FP) as a reporter gene driven by pro-inflammatory cytokine promoters such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNFα). In the reporter-transfected RAW264 cells, the protein expression levels of green fluorescence protein (GFP) were increased by inflammatory stimulations such as lipopolysaccharide (LPS), conditioned medium prepared using hypertrophied 3T3-L1 adipocytes, and a co-culture system. The changes in fluorescence intensity were equivalent to those of the mRNA and protein expression levels for each cytokine. Moreover, the effects of 15-deoxy-12,14Δ-prostaglandine J(2), a natural anti-inflammatory compound, were detectable in this system. These data indicate that the FP system developed here is an analysis system of low cost with simple procedures for evaluating inflammation, suggesting usability in the large-scale screening of anti-inflammatory compounds.
authors.affiliation: Laboratory for Molecular Functions of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Yuko
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Goto
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Teruo
authors.initials: T
authors.lastname: Kawada
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000032:analysis* / Q000494:pharmacology / Q000627:therapeutic use; D001681:Biological Assay; D018932:Chemokine CCL2 / Q000737:chemistry / Q000235:genetics*; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D007333:Insulin Resistance / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009765:Obesity / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics*
chemical_list: D000893:Anti-Inflammatory Agents; D018932:Chemokine CCL2; D017077:Culture Media, Conditioned; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins