SONODA Tadashige Oita Medical University, Dematology, Instructor, 医学部, 助手 (80244169)
ITAMI Satoshi Oita Medical University, Dermatology, Associate Professor, 医学部, 助教授 (30136791)
|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1993 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Intact sebaceous glands were isolated from full-thickness skin after incubation in dispase by using fine tweezers and dissecting microscope.
Isolated sebaceous gland lobules were placed on a 3I3-cell feeder layr in DMEM supplemented with 10% FCS.After 2-3wks dispersed cells were plated in 24 wells and maintained in KGM.Five strains (3 from face, 1 from nape and 1 from thigh) of cultured sebaceous cells were incubated with 25nM of H^3-testosterone for 4 hr at 37C.Metabolites were analyzed with thin-layr chromatography, high performance liquid chromatography and recrystallization.In four cases, the predominant metabolite was androstenedione, followed by androsterone, androstanedione and dihydrotestosterone.Thus, 17beta-oxidation was much more active than 5alpha-reduction.Preincubation with R1881, a synthetic androgen, did not affect such metabolic pattern.besides, culture with 10% FCS-DMEM,causing stratification of epidermal cells, did not affect the ratio of 5alpha-reduction to 17beta-oxidation, although much larger amounts of androstenedione and androstanedione were formed.In one case (obtained from nape), 5alpha-reduction was more active than 17beta-oxidation : a significant amount of dihydrotestosterone and 3alpha, 17beta-androstanediol was formed.Thus, only in this strain cultured cells maintain a characteristic pattern of metabolism of testosterone in vivo.
Apocrine glands were isolated from axillary skin under operation microscope, treated with trypsin and placed on a 3T3-cell feder layr in DMEM supplemented with 10% FCS.Subcultured cells were maintained in KGM.Contamination of fibroblasts was the major problem and has not been solved yet.Two strains of cultured cells were incubated with H^3-testosterone, as described above.
17beta-Oxidation predominated, as seen in cultured epidermal cells.Immunostaining with poyclonal anti-androgen receptor antibody NH-27 was negative both in cultured cells of sebaceous glands and apocrine glands.