A cell-based assay system for high-throughput screening of anti-wrinkle agents in human dermal fibroblast transfectant cells.
A cell-based assay system for monitoring COL1A2 [alpha2(I) collagen gene] promoter activity was developed to determine the influence of activated COL1A2 promoter in human dermal fibroblast cells. A pLuc-COL1A2 promoter plasmid that expresses the luciferase reporter gene in response to COL1A2 promoter activity was constructed. The pLuc-COL1A2 promoter plasmid and pCI-neo plasmid containing the NPT (neomycin phosphotransferase) gene for Geneticin resistance in host cells were co-transfected into human dermal fibroblast cells. COL1A2 promoter activities were measured by luciferase reporter gene assay using a luminescence detection method. Fibroblast cell transfectants treated with TNFalpha (tumour necrosis factor alpha), known to be an inhibitor of COL1A2 promoter expression, showed a reduction of COL1A2 promoter activity in a concentration-dependent manner, whereas TGF-beta (transforming growth factor-beta), known to be a stimulator of COL1A2 promoter expression, increased COL1A2 activity in a concentration-dependent manner. This assay system could be used to quantitatively measure COL1A2 promoter activity in human dermal fibroblast cells and allow the screening of anti-wrinkle agents from various synthetic chemicals and natural products.
Huh
Sungran
S
Biospectrum Life Science Institute, 101-701 SK Ventium, 522 Dangjung Dong, Gunpo City, 435-833 Gyunggi Do, Republic of Korea.
Lee
Jongsung
J
Jung
Eunsun
E
Ham
Yeonggeun
Y
Kim
Sang Suk
SS
Hyun
Chang Gu
CG
Kim
Yeong Shik
YS
Park
Deokhoon
D
D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D024042:Collagen Type I; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D015595:Skin Aging; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D024042:Collagen Type I; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen; D008156:Luciferases