Demonstration of downregulation of alpha-smooth muscle actin in interferon-gamma-treated myofibroblast by a novel cell-capture enzyme immunoassay.
We developed a simple method for determining the relative amount of alpha-smooth muscle actin (alphaSMA) produced in fibroblasts. The principle of the method is based on an enzyme immunoassay (EIA) for alphaSMA in microcultured fibroblasts. The optimized protocol of the assay is as follows. Human fibroblasts were cultured with transforming growth factor beta1 (TGFbeta1) in a microtiter plate and directly immobilized on the plate. The alphaSMA produced was labeled and subjected to indirect enzyme immunoassay using alkaline phosphatase, and optical density was measured. Semiquantitativeness was confirmed using various numbers of cells in which alphaSMA production was induced by treatment with TGFbeta1. The assay simply demonstrated that interferon-gamma (INF-gamma) inhibited the production of alphaSMA in an established cell line and that in primary cultured cells originated from the contractile nodule. Since the assay is simple and semi-quantitative, it is useful for elucidating the mechanism of contractile diseases and screening a large number of substances that have an inhibitory effect on the change in activity of myofibroblasts.
Tanaka
K
K
Department of Orthopedic Surgery, Osaka Medical College, Japan.
Sano
K
K
Tanaka
K
K
Kobayashi
M
M
Katsumura
K
K
Ikeda
T
T
Abe
M
M
D000199:Actins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D002460:Cell Line; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000737:chemistry*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000199:Actins; D016212:Transforming Growth Factor beta; D007371:Interferon-gamma