司坦唑醇对雌激素受抑的青春期大鼠生长板软骨细胞生长和成熟的影响

Effects of Stanozolol on Proliferation and Differentiation of Cultured Growth Plate Chondrocytes from Pubertal Female Rat Treated with GnRHa In Vitro

【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增殖和分化的影响。【方法】将6只经GnRHa处理后雌鼠的原代软骨细胞分为时效组(观察ST干预时限的影响)、量效组(观察ST干预剂量的影响)。时效组和量效组中,又分为ST干预时效组,对照时效组;ST干预量效组,对照量效组。通过噻唑兰比色分析法(MTT)法和免疫组化法检测软骨细胞核增殖细胞核抗原(PCNA)、胞浆中Ⅱ型胶原、Ⅹ型胶原的表达。【结果】(1)对MTT、PCNA的影响改变①时效组:ST作用后1天2参数已有显著升高(P<0.05),并与基值比较,第2天时达峰(P<0.001)。但第4下降至与0天时比较无差异(P>0.05)。②量效组:低浓度的ST无显著促增殖效应(P>0.05),而浓度增加至10-9mol/L时促增殖效应显现(P<0.01),进一步加大ST浓度至10-5mol/L,促增殖效应又开始减弱。(2)对软骨细胞胶原Ⅱ、Ⅹ合成的影响:①时效组:ST作用3d后胶原Ⅱ的表达明显高于对照组(P<0.05),但至第5天却表达显降。ST作用3d内胶原Ⅹ分泌量与对照组比较无差异,4d起显著递增(P<0.001)。②量效组:ST各个浓度点的软骨细胞胶原Ⅱ表达较基值均明显增加(P<0.01),并以10-9mol/L最显。10-11mol/L～10-8mol/L的ST不增加胶原Ⅹ表达,至10-7mol/L始随剂量增加而增,10-5mol/L时达最高值(P<0.01)。量、时效影响Ⅹ型胶原表达增加均迟于Ⅱ型,作用呈错峰性改变。【结论】ST以时效和量效作用方式分别对雌激素受抑的离体青春期大鼠生长板软骨细胞的增殖呈双相影响,在合适的剂量和时间时,细胞增殖效应可达最好效果。

[ Objective ] To observe the effect of stanozolol on proliferation and differentiation of cultured growth plate chondroeytes in vitro. [Methods] At 3 week of age, Sprague- Dawley rats received 2.5 mg/kg in slow-released GnRHa （triptorelin） which was repeated every 2 weeks for 2 times, at 7-week old. The tibial growth plate cartilage were aseptically dissected and tripsin and EDTA digested for 0.5 h, then collagenase digested for 3 h at 37 ℃. Chondrocytes were cultured in DMEM： F12 medium for 48 h, the cells were starved for 24 h in serum-free DMEM： F12 medium before stanozolol treatment. In dose-effect groups, chondroeytes were incubated in serum-free media in various concentrations of stanozolol for 48 h. In timecourse groups, chondrocytes were incubated in serum-free media in various times of stanozolol （10^-8 mol/L）, immunohistochemical staining of collagen Ⅱ , X , PCNA, and MTT were conducted. [Results] The results of MTT, PCNA, and type Ⅱ collagen synthesization demonstrated stanozolol enhanced the proliferation of the chondrocytes, time-course studies had shown that the proliferation were maximally stimulated by stanozolol after 2 or 3 days of incubation and decreased again after longer periods of incubation. Stanozolol stimulated the proliferation of the chondrocytes dose-dependently at 10^-11 mol/L and 10^-8 mol/L, maximally stimulatory concentrations of Stanozolol was 10^-9- 10^-8 mol/L, and decreased again after higher concentration of stanozolol. Stanozolol did not stimulated type X collagen synthesization from 10^-11 mol/L - 10^-8 mol/L, but experiments showed that type X collagen was already stimulated after incubation in Stanozolol （10^-7 - 10^-5 mol/L）. Time-course studies had shown those type X collagen synthesizations were stimulated by stanozolol after 4 - 5 days of incubation. [Conclusion] Stanozolol enhances the proliferation of chondrocytes of pubertal female rat treated with GnRHa in vitro （time-course- dependent and concentration -dependent）.