生长激素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增殖和表型的影响

Effect of growth hormone on proliferation and phenotype of cultured growth plate chondrocytes from pubertal rats treated with GnRHa

目的研究生长激素(GH)对促性腺激素释放激素拟似物(GnRHa)处理后的离体青春期大鼠生长板软骨细胞增殖和表型的影响。方法采用两步酶消化法分离培养经GnRHa处理后的5～8只雌鼠胫骨生长板软骨细胞,用四氮甲基唑蓝(MTT)、流式细胞仪测定细胞周期以及免疫组化法测定增殖细胞核抗原(PCNA)和胶原Ⅱ(ColⅡ)的表达,观察不同作用时间和不同剂量的GH对软骨细胞增殖和表型的影响。结果 GH能促进体外培养GnRHa处理后的青春期大鼠生长板软骨细胞的增殖,并存在一定的时效和量效关系。GH作用后第1天、第2天、第3天、第4天、第5天、第6天组与对照组比较有统计学意义(P<0.05),并以第4天时的促增殖效应最强。GH浓度低时(10ng/ml,20ng/ml)促增殖效应不明显(P>0.05),而浓度增加至50ng/ml时促增殖效应开始显现(P<0.05),至100ng/ml时促增殖效应最明显。GH亦能促进ColⅡ的表达,以GH作用后第4天,浓度为100ng/ml时ColⅡ的表达最强。结论 GH能以直接作用的方式促进体外培养的经GnRHa处理后的大鼠胫骨生长板软骨细胞的增殖和ColⅡ的表达,GH促增殖的剂量与临床应用剂量存在一致性,为GH克服GnRHa使用过程中所造成的生长减速提供了实验理论依据。

Objective To observe the effect of growth hormone on proliferation and phenotype of cultured growth plate chondrocytes from pubertal rats treated with GnRHa. Methods Primary chondrocytes from tibial growth plate of 5-8 female rats by trypsin-collagenase digestion were cultured in monolayer. MTT method,cell cycles detection by flow cytometry and immunohistochemical staining of PCNA and colⅡ were conducted to observe the influence of GH with different time and different concentrations on the proliferation and phenotype of chondrocytes cultured in vitro. Results GH enhanced the proliferation of chondrocytes of pubertal rats treated with GnRHa in vitro in time-dependent and concentration-dependent fashion. GH could significantly promote the proliferation of chondrocytes at day 1,day 2,day 3,day 4,day 5 and day 6,which had a significant difference compared with the control group （ P 〉0. 05）. The optimal time for proliferation occurred at day 4. The proliferation of chondrocytes was not obvious with GH concentrations of 10 ng/ml and 20 ng/ml （ P 〈0. 05）. The proliferative effect began to occur with GH concentration up to 50 ng/ml （ P 〈0. 05） and the optimal concentration was 100 ng/ml. GH also enhanced the expression of colⅡ. The optimal time for expression occurred at day 4 and the optimal concentration was 100 ng/ml. Conclusions GHenhances the proliferation and ColⅡexpression of chondrocytes from pubertal rats treated with GnRHa in vitro directly. The dose for proliferation-promotion is consistent with the dose administered in clinical practice. It provides a theoretical basis for overcoming deceleration of growth rate during GnRHa treatment.