Zhu Shun-ye, Li Yan-hong, Ma Hua-mei, Pan Si-nian, Chen Hong-shan, DU Min-lian
Department of Pediatrics, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510089, China.
Zhonghua Er Ke Za Zhi. 2009 Oct;47(10):774-8.
To investigate the effects and the mechanisms of stanozolol (ST) on the proliferation, maturation and differentiation of in vitro cultured growth plate chondrocyte isolated from gonadotropin releasing hormone analogue (GnRHa)-treated adolescent rats, to study if ST mediates the proliferation of chondrocytes via the estrogen receptor alpha (ERalpha), androgen receptor (AR) and/or insulin-like growth factor-1 receptor (IGF-1R) and interactions of the two receptor and IGF-1R receptor signaling pathway, to investigate the mechanism of the biological effects in ST promoting bone growth/maturity at molecular level.
The rats were weaned at the end of 3 weeks and intramuscular injection of triptorelin of GnRHa preparations, qow x 2 was started. The rats were sacrificed at the end of 7 weeks, and then the tibiae growth plates were taken out with sterile procedure. The chondrocytes were obtained by two-time enzyme digestion method, and the experiments were carried out with the primary chondrocytes. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and Western blot analysis were applied.
The results of PCNA demonstrated that stanozolol enhanced the proliferation of the chondrocytes, time-course studies showed that the proliferation were maximally stimulated by stanozolol after 2 days of incubation and decreased again after longer periods of incubation. The expression of p-ERalpha, p-IGF-1R and p-extracellular-signal regulated kinase 1/2 (ERK1/2) increased with the incubation period of ST treatment, and reached the peak value at a certain time, and then gradually decreased. The expression of p-ERalpha, p-IGF-1R and p-ERK1/2 increased with the elevation of ST concentration, and reached the peak value at 10(-9) - 10(-8) mol/L, then gradually decreased. ST induced-p-ERalpha expression was partially blocked by ERalpha and mitogen-activated protein kinase kinase inhibitors. ST induced-p-IGF-1R expression was partially blocked by ERalpha and IGF-1R inhibitors. ST induced-p-ERK1/2 expression was partially blocked by mitogen-activated protein kinase kinase and IGF-1R inhibitors.
As an androgen derivation, ST exerts its biological effects of promoting proliferation of the long bone growth plate chondrocytes via activating the classic ERalpha receptor pathway and mitogen-activated protein kinase pathway, and at the same time, by activation of IGF-1R. Both IGF-1R and ERalpha can promote "cross-talk" of two systems' receptor signal through mitogen-activated protein kinase signal pathway.
探讨司坦唑醇（ST）对促性腺激素释放激素类似物（GnRHa）处理的青春期大鼠体外培养生长板软骨细胞增殖、成熟及分化的影响及其机制，研究ST是否通过雌激素受体α（ERα）、雄激素受体（AR）和/或胰岛素样生长因子-1受体（IGF-1R）介导软骨细胞增殖以及两种受体与IGF-1R受体信号通路的相互作用，从分子水平探讨ST促进骨骼生长/成熟生物学效应的机制。
大鼠于3周龄末断奶，开始肌肉注射GnRHa制剂曲普瑞林，每周1次，共2次。7周龄末处死大鼠，无菌操作取出胫骨生长板。采用二次酶消化法获取软骨细胞，以原代软骨细胞进行实验。应用增殖细胞核抗原（PCNA）免疫组化染色及蛋白质印迹分析。
PCNA结果显示，司坦唑醇增强软骨细胞增殖，时间进程研究表明，培养2天后司坦唑醇对增殖的刺激作用最大，培养更长时间后作用减弱。p-ERα、p-IGF-1R和p-细胞外信号调节激酶1/2（ERK1/2）的表达随司坦唑醇处理培养时间延长而增加，在某一时刻达到峰值，然后逐渐下降。p-ERα、p-IGF-1R和p-ERK1/2 的表达随司坦唑醇浓度升高而增加，在10⁻⁹ - 10⁻⁸ mol/L时达到峰值，然后逐渐下降。ERα和丝裂原活化蛋白激酶激酶抑制剂可部分阻断司坦唑醇诱导的p-ERα表达。ERα和IGF-1R抑制剂可部分阻断司坦唑醇诱导的p-IGF-1R表达。丝裂原活化蛋白激酶激酶和IGF-1R抑制剂可部分阻断司坦唑醇诱导的p-ERK1/2表达。
作为一种雄激素衍生物, 司坦唑醇通过激活经典的ERα受体途径和丝裂原活化蛋白激酶途径发挥促进长骨生长板软骨细胞增殖的生物学效应，同时通过激活IGF-1R发挥作用。IGF-1R和ERα均可通过丝裂原活化蛋白激酶信号通路促进两个系统受体信号的“串话作用”。
