睾酮通过上调血管内皮生长因子表达促进外周血内皮祖细胞增殖

Testosterone Enhances the Proliferation of Peripheral-Blood-Derived Endothelial Progenitor Cells by up-regulating Vascular Endothelial Growth Factor Expression

目的探讨雄激素对外周血内皮祖细胞(PB-EPC,)增殖能力的影响及其可能机制。方法将健康志愿者外周血经密度梯度离心法分离的单个核细胞接种至人纤维连接蛋白包被的培养板中,EGM-2MV培养7天后,多波长激光共聚焦显微镜鉴定FITC标记的荆豆凝集素和DiI标记的乙酰化低密度脂蛋白双染色阳性为PB-EPC。将贴壁细胞分为5组,前4组分别加入0、1、10、100 nmol/L睾酮,第5组加入10 nmol/L雄激素受体阻断剂氟他胺干预3 h后,再加10 nmol/L睾酮干预。培养48 h后,MTT比色法检测各组PB-EPC的增殖能力。实时定量PCR检测血管内皮生长因子(VEGF)mRNA的表达变化,ELISA检测VEGF分泌量的变化。结果睾酮呈浓度依赖性促进EPC增殖,雄激素受体阻断剂氟他胺完全阻断睾酮对EPC的促进作用。与空白对照组相比,睾酮在mRNA和蛋白水平上调PB-EPC的VEGF表达,氟他胺可阻断此作用。结论睾酮通过雄激素受体途径上调VEGF的表达,促进PB-EPC增殖。

Aim To explore the effects and related mechanisms of testosterone on the proliferation of Peripheral-blood endothelial progenitor cells （PB-EPCs）. Methods Total mononuclear cells（MNC） were isolated from peripheral blood of healthy volunteers by Ficoll density gradient centrifugation, culturing with EGM-2MV for 7 days in vitro. The adherent cells showed up taking of acetylated low-density （ac-LDL-DiI） and binding of lectin（FITC-UEA-I）, observing with confocal laser scanning microscopy. PB-EPC were dealt with four concentrations of testosterone, as 0 nmol/L, 1 nmol/L, 10 nmol/L,and 100 nmol/L respectively, and in another group PB-EPC were pretreated with 10 nmol/L flutamide （androgen receptor antagonist） for 3h and then stimulated with 10 nmol/L testosterone. After 48 h, the ability of cell proliferation was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diph-phenyltetrazolium bromide assay （MTT）. The VEGF expression was tested by quantitative real-time RT-PCR and ELISA assay. Results The proliferation of PB-EPC were increased by testosterone in a dose-dependent manner, however, these effects could be blocked by flutamide. Testosterone can up-regulate VEGF both on mRNA and protein level, however, these effects could be blocked by flutamide. Conclusions Testosterone enhances the proliferation of PB-EPCs by up-regulating VEGF expression via androgen receptor pathway.