1990 Fiscal Year Final Research Report Summary
Molecular Biological Study on Mechanism of Blister Formaion in Pemphigus
| Project/Area Number |
01570566
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Dermatology
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| Research Institution | Osaka University School of Medicne |
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Principal Investigator |
HASHIMOTO KOJI Osaka University School of Medicine Associate Professor, 医学部, 助教授 (00110784)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Urokinase / PAI-1 / Pemphigus / TGF-beta / Epidermal Keratinocyte / Anti-mouse urokinase antibody / mRNA / Northerna Blot |
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| Research Abstract |
Effects of pemphigus antibody on induction of urokinase (uPA) mRNA in cultured human keratinocytes were examined. After addition of purified IgG from pemphigus patients to culture medium containing 1.8 mM Ca, total RNA was purified and uPA mRNA was analyzed by Northern blot. Addition of pemphigus vulgaris IgG (8mg/ml) induced marked increase of uPA mRNA compared to normal IgG at 3 h. Next, dose-dependency of uPA mRNA induction by pemphigus IgG was examined. Human keratimocytes were incubated with pemphigus vulgaris IgG from two patients up to 4 and 8 mg/ml, respectively, for 3 h. The amount of uPA mRNA was increased dependent on pemphigus IgG concentration, being 2.5 fold at 4 mg/ml and 4.5 fold at 8 mg/ml. This result indicates that the increase of uPA activity in human keratinocytes by pemphigus IgG was accompanied by induction of uPA mRNA. Detection of uPA mRNA was performed using total RNA purified from neonatal mouse skin. RNA from whole epidermis of one neonatal mouse is enough to detect mouse uPA mRNA.
Purification of plasminogen activator inhibitor-1 (PAI-1) was performed to examine whether PAI-1 inhibits blister formation by pemphigus IgG in neonatal mouse. PAI-1 was purified from 2 liter of culture medium incubated with TGF-betal (5 ng/ml) by Con A column and affinity chromatography. Finally, 500mug of PAI-1 was obtained. However, inhibitory activity of purified PAI-1 was markedly decreased by SDS-treatment for activation. Therefore, neonatal experiment could not be achieved.
Mouse urokinase was purified from culture medium of PAM cell. Anti-mouse urokinase antibody was raised. Neutralizing activity of this antibody is now being examined.
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