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

人皮脂腺和顶浆腺培养细胞的雄激素代谢

• 批准号: 05670736
• 负责人: TAKAYASU Susumu
• 金额: $ 1.34万
• 依托单位: Oita Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670736/
• 关键词: Sebaceous gland; Apocrine gland; Culture; Androgen metabolism; 男性ホルモン受容体; 5alpha-レダクターゼ; 培養細胞

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.

用细镊子和解剖显微镜从全层皮肤上分离出完整的皮脂腺。将分离的皮脂腺小叶置于添加10% FCS的DMEM中的3i3细胞饲养层上。2-3周后，将分散的细胞置于24孔中，并在KGM中保存。将培养的5株皮脂腺细胞（3株面部、1株颈背、1株大腿）与25nM的H^3-睾酮溶液在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物。4例主要代谢物为雄烯二酮，其次为雄酮、雄烯二酮和二氢睾酮。因此，17 -氧化比5 -还原更活跃。与R1881（一种合成雄激素）预先孵育不影响这种代谢模式。另外，10% FCS-DMEM培养引起表皮细胞分层，但不影响5 α -还原与17 β -氧化的比例，但形成的雄烯二酮和雄烯二酮数量要大得多。在一种情况下（从颈背中获得），5 - α还原比17 - β氧化更活跃：形成了大量的双氢睾酮和3 - α， 17 - β -雄甾二醇。因此，只有在该菌株中培养的细胞维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大汗腺，胰蛋白酶处理后置于3t3细胞饲料层中，添加10% FCS。传代培养细胞保存在KGM中。成纤维细胞的污染是主要问题，但尚未得到解决。如上所述，两株培养细胞与H^3-睾酮孵育。在培养的表皮细胞中，β -氧化占主导地位。培养的皮脂腺和大汗腺细胞用抗雄激素受体抗体NH-27免疫染色均为阴性。