Androgen metabolism by cultured cells derived from human sebaceous gland and apocrine gland

将分离的皮脂腺小叶置于含10%FCS的DMEM培养液中的3 I3细胞饲养层上，2- 3周后将细胞分散于24个威尔斯孔中，并在KGM中培养。将培养的皮脂腺细胞（3个来自面部，1个来自颈背，1个来自大腿）与25 nM的H^3-睾酮在37 ℃下孵育4小时。用薄层色谱法、高效液相色谱法和重结晶法分析代谢产物。在4个病例中，结果表明，在体外培养的细胞中，17 β-氧化代谢的活性明显高于5 α-还原代谢，而在10% FCS-DMEM中培养的细胞中，5 α-还原代谢与17 β-氧化代谢的比值不受影响，尽管形成了大量的雄烯二酮和雄甾烷二酮。（得自nape），5 α-还原比17 β-氧化更有活性：形成了大量的二氢睾酮和3 α，17 β-雄甾烷酮。因此，只有在该菌株中培养的细胞保持体内睾酮代谢的特征模式。在手术显微镜下从腋窝皮肤分离顶泌腺，用胰蛋白酶处理并置于含10%FCS的DMEM中的3 T3细胞饲养层上。传代培养的细胞在KGM中维持。成纤维细胞的污染是主要问题，尚未解决。如上所述，将两种培养细胞与H^3-睾酮孵育。17 β-氧化占主导地位，用多环抗雄激素受体抗体NH-27的免疫染色在皮脂腺和顶泌腺的培养细胞中均为阴性。

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.