雌性激素对骺生长板软骨细胞周期调节蛋白的作用

Effect of estrogen on chondrocyte cyclin in epiphyseal growth plate

背景:长骨干骺端生长板是哺乳动物在生长发育期间实现线性生长的结构基础。激素及生长因子对生长板软骨细胞的调节作用与生长、生长板老化直至完全闭合具有决定性意义。目的:观察雌性激素对骺生长板软骨细胞周期蛋白表达的影响,及其在生长板闭合过程中的作用。设计、时间及地点:随机对照动物实验,于2003-07/2005-07在重庆医科大学动物实验中心完成。材料:健康雌性12周龄新西兰白兔30只,平均体质量1.57kg。方法:30只新西兰白兔性发育前(12周龄)行双侧卵巢切除,4周后(16周龄)随机抽签法分为2组:雌二醇组每周苯甲酸雌二醇肌肉注射,140μg/kg;对照组:每周不含雌二醇的等体积灭菌棉籽油肌肉注射(1.5mL/kg)。两组动物分别于处理后第4,7,10周(20,23,26周龄)取双侧股骨远端、胫骨近端干骺部分生长板作免疫组织化学染色。主要观察指标:两组动物不同年龄股骨和胫骨未闭合骺生长板软骨细胞细胞周期蛋白D1、P53、P21WAF1/CIP1及P16INK4A蛋白的表达。结果:①雌二醇组雌兔实验后4周相当于性发育早期(20周龄),胫骨近端、股骨远端骺生长板细胞周期蛋白D1表达水平显著高于同年龄期对照组(P<0.05);7周后(23周龄)胫骨近端骺生长板细胞周期蛋白D1表达仍高于对照组雌兔(P<0.05),股骨远端骺生长板细胞周期蛋白D1表达开始降低,与对照组雌兔无显著差异(P>0.05)。②实验后4,7周(20～23周龄)雌二醇组雌兔股骨远端和胫骨近端骺生长板软骨细胞P53蛋白表达明显高于对照组(P<0.05)。③雌二醇组P21WAF1/CIP1的表达增高则出现在实验后7周(23周龄),与对照组相比,差异有显著性意义(P<0.05)。④两组雌兔骺生长板P16INK4A蛋白免疫组织化学检测均为阴性。结论:雌二醇具有加速生长板闭合过程的作用,可能与雌性激素长期作用于软骨细胞后诱导细胞周期抑制蛋白如P21WAF1/CIP1、P53有关。

BACKGROUND： The growth plate in the metaphyseal of long bones is the structural basis for linear growth factor exhibit an important role in regulation the growth, senescence and fusion process of the growth plate. OBJECTIVE： To explore the effect of estrogen on chondrocyte cyclin in epiphyseal growth plate, in addition fusion process of the growth plate. Estrogen and growth on the function of DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Chongqing Medical University between July 2003 and July 2005. MATERIALS： A total of 30 New Zealand rabbits, with 12-weeks-old, weighing 1.57 kg were selected. METHODS： All rabbits were divided into the estradiol and control groups by method of random sampling at 4 weeks after ovariectomy. The rabbits in the estradiol group were given intramuscular injection of estradiol （140 μg/kg, once per week）; the control group was injected matching sterile cotton oil without estradiol. A part of growth plate of bilateral distal femurs and proximal tibia were obtained for immunohistochemistry staining at weeks 4, 7, and 10 after injection. MAIN OUTCOME MEASURES： The expression of cyclin DI, P53, e2twAF1/CIP1 and protein P16INK4A in growth plate distal femur and proximal tibia was observed. RESULTS： The Cyclin D~ expression both in the distal femur and proximal tibia growth plate chondrocytes were obvious increased in the estradiol group at early sexual development （20 weeks old） than the control group （P 〈 0.05）, then, proximal tibia growth plate chondrocytes was remained higher than the control group at 7 weeks （23 weeks old, P 〈 0.05）, but the cyclin D~ expression in the distal femur was declined, which has no significant differences （P〉 0.05）. The P53 expression of the chondrocytes in the estradiol group was higher than the control group in both distal femur and proximal tibia at weeks 4 and 7 （P 〈 0.05）. The expression level of P21WAF1/CIP1 was increased in the estradiol group at 7 weeks than that of the control group, the difference between them was significant （P 〈 0.05）. More over, the P16INK4A protein expression was negative in epiphyseal growth plates. CONCLUSION： Estradiol can accelerate the processes of growth plate fusion, which maybe related to the effect of estrogen on induction the expression of cell cycle control protein, such as P21WAF1/CIP1 and P53.