睾酮对血管平滑肌细胞中雄激素受体蛋白表达调控的研究  

Testosterone regulation of androgen receptor protein expression in cultured vascular smooth muscle cells

作　　者：马瑞[1] 吴赛珠[1] 林其谁[2] 

机构地区：[1]第一军医大学南方医院心血管内科,广州510515 [2]中国科学院上海生物化学与细胞生物学研究所分子生物学国家重点实验室

出　　处：《中华医学杂志》2004年第6期491-495,共5页National Medical Journal of China

基　　金：国家"九七三"重点基础研究发展计划资助项目(G2 0 0 0 0 5 70 0 8)

摘　　要：目的　观察睾酮对雄性大鼠血管平滑肌细胞中雄激素受体 (AR)蛋白表达的调控作用 ,并对其调控水平作初步探讨。方法　贴块法培养雄性SD大鼠胸主动脉血管平滑肌细胞 ,分别用细胞裂解和高盐提取法制备细胞质和细胞核抽提物 ,Western印迹法检测提取物中AR水平。结果　不同浓度 (0～ 4 μmol/L)睾酮与静止的血管平滑肌细胞作用 2 4h呈剂量依赖性地增加胞质和胞核内AR蛋白含量 ,生理水平 (40nmol/L)睾酮与细胞作用 10min引起胞质内AR蛋白水平的快速下调 ,继而呈时间依赖性 (1～ 4 8h)地增加胞质和胞核内AR蛋白表达 ;预先给以转录抑制剂更生霉素和翻译抑制剂cycloheximide则能使上调的胞质内AR蛋白分别降至单纯睾酮刺激组水平的 4 6 %和 12 % ;雄激素拮抗剂氟他胺也能够部分抑制 (5 0 % )睾酮对细胞内AR蛋白水平的上调作用。结论　血管平滑肌细胞中存在睾酮对AR蛋白表达的自身上调作用 ,其中伴随着受体的核转位 ;调控过程既有转录机制、也有转录后机制的参与 ,并且需要功能性AR的介导。Objective To investigate the effects of testosterone exposure on androgen receptor (AR) protein expression in vascular smooth muscle cells (VSMC) and the possible mechanisms mediating the effects. Methods VSMC were cultured from thoracic aorta of male Sprague-Dawley rats by using the explant method. Cytoplasmic and nuclear extracts were prepared by means of cell lysis and high salt extraction respectively, and subjected to western blotting analysis for determination of AR protein level. Results Treatment of synchronized VSMC with testosterone increased both cytoplasmic and nuclear AR protein expression in a dose (0～4 μmol/L) and time (1～48 h) -dependent fashion, whereas exposure of VSMC to testosterone at a physiologically relevant concentration of 40 nmol/L for 10 min induced a transient down-regulation of AR protein. Pretreatment with transcription inhibitor actinomycin D and translation inhibitor cycloheximide repressed cytoplasmic AR protein leves to 46% and 12% (means) of the androgen treatment control level respectively. Furthermore, androgen up-regulation of intracellular AR protein was partially inhibited (50%) by nonsteroidal androgen antagonist, flutamide. Conclusion Homologous up-regulation of AR protein expression exist in synchronized VSMC, and the auto-regulation is time and testosterone dose dependent, accompanied by nuclear translocation of AR protein, and requires functional AR protein. In addition, our present data suggest that testosterone increases AR protein expression in VSMC at the level of both transcription and translation.

关 键 词：睾酮 血管平滑肌细胞 雄激素受体 蛋白表达 WESTERN印迹法 冠心病 动脉粥样硬化 

分 类 号：R541.4[医药卫生—心血管疾病]