摘要
目的探讨司坦唑醇（ST）对离体培养的促性腺激素释放激素拟似物（GnRHa）处理后青春期大鼠生长板软骨细胞的作用及其分子层面机制。方法设计并处理后获得胫骨原代软骨细胞，采用免疫组织化学法，检测ST处理后软骨细胞核增殖细胞核抗原（PCNA）的表达；采用Western Blot法，分析ST作用后软骨细胞雌激素受体α（ERa）、雄激素受体（AR）、胰岛素样生长因子1受体（IGF-1R）及细胞外信号调节激酶（ERK1／2）表达的改变。结果（1）ST处理后，PCNA呈阳性的细胞明显增加，与0d组（0．05％）比较，1d、2d、3d组的阳性率差值显著增加（分别为11．0％，24．0％，14．0％）。（2）延长ST作用时间和增加ST浓度，AR均未能被激活。ST作用后磷酸化（P）．ERa、P—IGF-1R、P-ERK1／2表达上调，并存在时间和浓度依赖；ERa或丝裂原激活蛋白激酶激酶（MAPK）被阻断后，ST所致的P—ERa表达较未阻断时减弱（1．18±0．07、2．35±0．05；1．45±0．17、2．77±0．39，P均〈0．05）；ERa或IGF-1R阻断后的p-IGF-1R较ST组显著减弱（4．42±0．42、8．00±0．30；0．77±0．17、11．37±0．97，P均〈0．05）；MAPKK或IGF．1R被阻断后p-ERK1／2较未阻断组显著减弱（0．61±0．14、9．26±0．92；4．27±0．76、8．59±0．52，P均〈0．05）。结论ST可以促进软骨细胞增殖，其作用主要经ERa介导，包括了经典的与ERa以配体结合方式和以非配体激活的MAPK途径，并同时激活IGF-1R，实现其促长骨生长板软骨细胞生长和成熟的生物效应。
Objective 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 α （ ERα）, androgen receptor （AR） and/or insulinlike 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. Method 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 chandrocytes. Immunohistochemical staining of proliferating cell nuclear antigen （PCNA） and Western blot analysis were applied. Result 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-ERos, 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-ERa, p-IGF-1R and p-ERKI/2 increased with the elevation of ST concentration, and reached the peak value at 10-9-10-Smol/L, then gradually decreased. ST induced-p-ERα expression was partially blocked by ERα and mitogen-activated protein kinase kinase inhibitors. ST induced-p-IGF-1R expression was partially blocked by ERα and IGF-1R inhibitors. ST induced-p-ERK1/2
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2009年第10期774-778,共5页
Chinese Journal of Pediatrics
