司坦唑醇对促性腺激素释放激素拟似物处理后的青春期大鼠生长板软骨细胞IGF-1 mRNA表达及其蛋白合成、分泌的影响

目的研究司坦唑醇（stanozolol，ST）对促性腺激素释放激素拟似物（gonadotropin releasing hormone agonist，GnRHa）处理后的离体青春期大鼠生长板软骨细胞胰岛素样生长因子-1（insulin-like growth factor-1，IGF-1）mRNA表达及其蛋白合成、分泌的影响。方法将6只雌鼠的原代软骨细胞，分为时效组、量效组。根据是否用司坦唑醇干预，将时效组和量效组分为时效干预组，时效对照组；量效于预组，量效对照组，分别观察司坦唑醇的干预时限和剂量对促性腺激素释放激素拟似物处理后离体青春期大鼠生长板软骨细胞增殖的影响。采用软骨细胞增殖能力测定法（MTT）和免疫组化法检测不同剂量、不同时间点司坦唑醇处理的离体的青春期大鼠生长板软骨细胞的增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）表达，荧光实时定量RT—PCR检测软骨细胞的IGF-1 mRNA表达。酶联免疫吸附法（enzyme—linked immunosorbent assay，ELISA）测定胰岛素样生长因子-1的合成。结果司坦唑醇以时效和量效作用方式，对雌激素受抑的离体青春期大鼠生长板软骨细胞增殖呈双相型影响。在合适的剂量和疗程时，软骨细胞增殖效应可达最好效果。①司坦唑醇作用2h后，软骨细胞IGF-1 mRNA表达显著增加，与时效对照组基础值相比，差异有显著意义（P〈0．05），并存在较强的时间依赖性；作用8h时，达最高峰（为时效对照组基础值的3．8倍）。司坦唑醇的作用在（10^-10～10^-5）mol／L时，与软骨细胞的细胞核抗原增殖效应存在明显的剂量依赖性（组间比较，差异有显著意义，P〈0．05）；在10^-7mol／L时，软骨细胞的增殖细胞核抗原表达达最高峰（为基础值的5．75倍）。②司坦唑醇作用自5h起，软骨细胞胰岛素样生长因子-1蛋白合成与时间点为0组比较，差异有显著意义（P=0．042）；5h-20h时，作用存在显著时间依赖性（P〈0．05）；作用20h时，达峰值（为量效对照组基础值的3．3倍）。与量效对照组比较，司坦唑醇自10^-10mol／L组，胰岛素样生长因子-1蛋白含量开始增加，并在10^-7mol／L组达峰值（P=0．000）。结论司坦唑醇可以量效、时效的作用方式，影响和增加对促性腺激素释放激素拟似物处理后的体外培养雌激素水平，促进青春期大鼠生长板软骨细胞IGF-1 mRNA的表达和胰岛素样生长因子-1的合成、分泌，推测司坦唑醇促进生长效应机制与生长板局部胰岛素样生长因子-1的自分泌、旁分泌增加有关。

Effects of Sta:nozolol on Local Insulin-Like Growth Factor-1 Synthesis(Insulin-Like Growth Factor-1 Peptide and Insulin-Like Growth Factor-1 mRNA Levels) in Cultured Growth Plate Chondrocytes

Objective To observe effects of stanozolol on proliferation and differentiation of cultured growth plate chondrocytes in vitro, and study whether local insulin-like growth factor-1 （IGF-1） synthesis （insulin-like growth factor-1 peptide and IGF-1 mRNA levels） in cultured growth plate chondrocytes were regulated by stanozolol. Methods Primary chondrocytes from 6 female rats were divided into 2 groups （time effect groups and concentration-effect groups）. Immunohistochemical staining of proliferating cell nuclear antigen （PCNA） and MTT were conducted. Primary passage cells were cultured in 35 mm plastic dishes for determination of local insulin-like growth factor-1 synthesis （ELISA） and mRNA （real-time RTPCR） extraction. Results The results of MTT, proliferating cell nuclear antigen demonstrated stanozolol enhanced the proliferation of the chondrocytes, time-course studies had shown that the proliferation were maximally stimulated by stanozolol after 2 days or 3 days of incubation and decreased again after longer periods of incubation. Stanozolol stimulated the proliferation of the chondrocyte dose dependently at 10^-11 mol/L and 10^-8 mol/L, maximally stimulatory concentrations of stanozolol was （10^-9 ～10^-8）mol/L. The expression levels of insulin-like growth factor-1 mRNA（insulin-like growth factor-1 peptide levels） by stanozolol-stimulated synthesis was dose-dependently at 10^-11 mol/L and 10^-7mol/L, maximally stimulatory concentrations of stanozolol was 10^-7 mol/L. Incubation with stanozolol 10^-7 mol/L increased expression levels of insulin-like growth factor-1 mRNA（for 8 h） in the chondrocytes 3.8-fold and insulin-like growth factor-1 peptide（for 20 h）in the supernatant 3.3-fold, respectively, compared to the solvent control group. Conclusion Stanozolol enhances the proliferation of chondrocytes of pubertal female rat who were treated with gonadotropin releasing hormone agonist in vitro （time-course-dependent and concentration-dependent）. Stanozolol can enhance local insulin-like growth factor-1 synthesis （insulin-like growth factor-1 peptide and IGF-1 mRNA levels） in cultured growth plate chondrocytes, endocrinal growth hormone/insulin-like growth factor-1 axis function are unaffected by stanozolol administration in pubertal female rat treated with gonadotropin releasing hormone agonist.