大鼠成骨细胞增殖及护骨素蛋白含量与甲状旁腺激素的调节效应

Effects of parathyroid hormone on proliferation of rat osteoblasts and osteoprotegerin expression

目的:体外观察甲状旁腺激素对大鼠成骨细胞增殖和护骨素分泌的调节作用。方法:实验于2005-06/2006-05在四川大学华西医院生物治疗国家重点实验室干细胞与组织工程研究室完成。采用酶消化法和骨组织块法联合分离培养新生SD大鼠颅骨成骨细胞,间歇加药方法将不同浓度0,10-6,10-7,10-8,10-9mol/L甲状旁腺激素作用于大鼠成骨细胞(0mol/L作为空白对照组),经碱性磷酸酶染色及钙结节茜素红染色证实培养的成骨细胞后,采用四甲基偶氮唑盐比色法检测细胞增殖能力,蛋白免疫印迹测定护骨素的分泌量。结果:①成骨细胞的形态变化:倒置相差显微镜下可见培养的成骨细胞呈短梭形、三角形和多边形。碱性磷酸酶染色光镜下可见成骨细胞胞浆中分布特征性蓝紫色细颗粒;成骨细胞的钙结节在镜下可见部分细胞聚集一起呈"集落状"生长。②成骨细胞增殖率:10-6～10-9mol/L浓度甲状旁腺激素对成骨细胞增殖均显示刺激作用,与空白对照组相比,差异显著(P<0.05),增殖率以10-8mol/L甲状旁腺激素组最高。③成骨细胞护骨素的表达:甲状旁腺激素下调成骨细胞中护骨素的分泌,与空白对照组相比,10-6mol/L甲状旁腺激素组抑制作用最明显(P<0.01),无显著剂量依赖性。结论:甲状旁腺激素对体外培养成骨细胞的增殖具有明显促进作用,通过下调成骨细胞中护骨素的分泌,促进破骨细胞生成、活化,促进骨吸收。

AIM： To investigate the effect of parathyroid hormone （PTH） on adjusting the proliferation and osteoprotegerin （OPG） expression of rat osteoblasts in vitro. METHODS： This study was carried Put in the Division of Stem Cell and Tissue Engineering, State Key Laboratory of Bio-Therapy of Sichuan University West China Hospital from June 2005＇to May 2006. Osteoblasts were isolated from the calvariae of newborn SD rats by combined means of enzymatic digestion and bone tissue block, and then given different concentrations of PTH in intermission （0, 10^-6, 10^-7 10^-8 and 10^-9 mol/L, 0 mol/L PTH as control）, The cultured osteoblasts were identified by the staining of alkaline phosphatase and calcium nodus alizarin red. The effect of PTH on proliferation of osteoblasts was observed by the tetrazolium salt test （MTT）, as well as the expression of OPG proteins were measured by protein immunoblotting. RESULTS： （1）Morphologic change of osteoblasts： The cultured osteoblasts, which were observed under inverted phase contrast microscope, had typical shapes （short fusiform） and structures （triangle and polygon）. The typical blue-violet drops were seen in osteoblasts plasma after alkaline phosphatase staining and calcium nodus were formed in colony.（2）Proliferation rate of osteoblasts： PTH in the concentration from 10^-6 mol/L to 10^-9 mol/L could enhance the proliferation of osteoblasts, the value of MTT was significantly increased compared with the controls （P 〈 0.05）, especially at 10^-8 mol/L. （3）Expression of OPG： PTH could remarkably down-regulated the expression of OPG compared with the controls, especially at 10^-6 mol/L （P 〈 0.01 ）, without a concentration dependent manner. CONCLUSION： PTH has obvious promoting effect on proliferation of osteoblasts cultured in vitro. PTH can promote the bone resorption by decreasing the expression of OPG in osteoblasts, and then stimulate the osteoclasts differentiation and activity.