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101

CULTURE OF KERATINOCYTES ON ARTIFICIAL SKIN DERMIS (COLLAGEN SPONGE) Y. MARUGUCHI’, T. MARUGUCHI’. S. SUZUKI’, N. ISSHIKI’ AND K. TODA3, ‘Department of Dermatology,

Kobe City General Hospital. Chuou-ku, Minatojima. Kobe. ‘Department of Plastic and Reconstructive Surgery. ‘Department of Dermatology, Faculty of Medicine. Kyoto University, Kyoto, Japan We previously developed a bilayer artificial skin composed

of outer silicone layer and inner collagen sponge, and then reported studies on the graft of this artificial skin. Recently we succeeded in culturing keratinocytes on this artificial skin dermis (ASD) with serum free medium. Three types of matrix were used; ASD coated with type I collagen gel. ASD coated with Matrigel, and ASD containing fibroblasts. Keratinocytes proliferated and differentiated on these matrix, and formed continuous cell layers on ASD. About 20 cell layers were obtained on Matrigel coated-ASD.

103

ICHTHYOSIS: A HISTOCHEMICAL AND Ih%llJNOHISTOCHEMICAL SllJDY

T. M4RUYAMA’. A.P.M. LAVRIJSEN’. M. PONEC’ and M. MOROHASHI1, ‘Department

of Dermatology. Tarama medical and Pharmaceutical University. and

ZDepartment of Dermatology, University Hospital L&den, The Netherlands

Expression of specific differentiation-protein markers has been studied in various forms of ichthyosis: ichthyosis vulgar-is, X-linked ichthrasis and lammelar ichtlwosis. For this purpose, antibodies recognizing keratin 1(1(l), keratin 10 (KlO) keratin 6 (K61, filaggrin [FL), transglutaminase ITG) and involucrin (IV) have been used. Furthermore.DACM stainings were used as a specific marker to SH groups and SS linkages in cellular proteins.

Xl and KIO expression in all cases studied was similer to that found in normal epidermis. K6, usually not present in normal epidermis was slightly to moderately expressed in all types of ichthyosis studied. Although the earlier expression of IV was found, there was no significant difference in TG expression between normal and ichthyotic skin, with the exception of one case of lammelar ichthyosis. The earlier expression of FL was found and was not related to the absence, presence or thickness of stratum granulosum. In contrast to the earlier appearance of membrane- located SH groups in the upper pricle layer. the localization of SS linkages was normal. These findings suggest that the disorders studied show signs of hyperproliferation and disregulation in the process of the cross-linked envelop formation.

105 INTERPHASE CYTOGENETICS OF MALIGNANT MELANOMAS, DYSPLASTIC NEVI. AND SPITZ NEVI : NUMERICAL ABERRATIONS OF CHROMOSOME 17 IN INTERPHASE NUCLEI.

M.MATSUTA’. Y.IMAMURA. S.KON AND J GRAY‘, ‘Department of Dermatoloqv. lwate Medical Un~verc~tv School of Medlclne. 19. 1 Uch!maru.etiorloka. Japan, and ‘Department of Laboratory Medlcme. Umverclty of Callforma San Franctsco. San Franc~sco. California. USA.

Ulng DNA probes speclflc for chromosome 17 per~centromere. we appl,ed ,n s,tu hybrlduatlon (ISHI fechmque fo rouflnely processed paraffin embedded tissue sect!ons of ten cases of pr~marv malignant melanomas.five cases of dysplastic nevi. and five cases of Spitz nev~. Tumor cells of malignant melanomas showed numerical aberrations from one to SIX ISH signals m comparison with one or two Ish signals of target sites of chromosome 17. Dvsplastlc nevus ceils showed numerlca! aberrations from one to four ISH signals of Spitz nevus cells. noimal epldermal cells and lymphocytes on the same sections. Further we showed the means of spots/nuclus (S/N) : 2.04 in tumor cells ‘of malignant melanomas, 1 5 I” dysplastic news cells. and 1.28 I” sp,tz nevus cells. We thus showed directly the cytogenetx abnormalltles of twnors. precursor lessons, and benign nev~ of malIgnant lessons on the routmely processed paraffin embedded tissue sectwns Inferphase cytogenetlcs by usmg routmely processed paraffin embedded tissue sectIons may prowde new dtagnostic creiterla to dlscrlmlnate between normal and abnormal cells at even early stage of tumorgenesls.

104

EXPRESSION OF HUMAN STEROID 5-ALPHA REDUCTASE IN ESCHERICHIA COLI.

C.MATSUI and M.MOROHASHI, Department of Dermatology, Faculty of Medicine, Toyama Medical and Pharmacreutical University,2630,Sugitani,Toyama-shi, Toyama, Japan.

The enzyme steroid 512 redutase that catalyzes the conversion of testosterone to dihydro- -testosterone, plays a central role in human sexual differentiation and androgen physiology. We tried to express the human 5ureductase cDNA in Esherichea coli. as the fusion protein that could be purified by affinity chromatography. The hybrid protein had the assumable molecular weight (71kDa) on SDS-PAGE and immunoblotting, though it was degradated. The serum of rabbit that was immunized by the purified hybrid protein, could detect the single band on immunoblotting of human prostate extracts. Immunohistochemical analysis of prostate and skin by this serum is now in progress.