体外培养成骨细胞成纤维生长因子23表达及其对激素的反应

 目的 研究体外培养大鼠成骨细胞在不同分化阶段的成纤维生长因子23（fibroblast growth factor 23,FGF23）基因表达水平,并观察其对钙、磷、甲状旁腺激素（PTH）和1,25二羟维生素D［1,25(OH)₂D₃］的反应。方法 酶消化法分离培养新生SD大鼠头盖骨成骨细胞,于细胞半汇合、汇合、基质堆积和矿化4个阶段收获细胞,Trizol一步法提取总RNA,Real-time PCR法检测其FGF23 mRNA水平,并通过其碱性磷酸酶（alkaline phosphatase,ALP）、骨钙素（bone gla-protein,BGP）和骨桥蛋白（osteopontin,OPN）的mRNA表达水平变化评价成骨细胞分化状态。进一步取汇合细胞,分别用3.2 mmol/L氯化钙、4.4 mmol/L β甘油磷酸钠（βGP）、10^-9 mol/L rhPTH(1-34)和10^-8 mol/L 1,25(OH)₂D₃作用3 d,检测其FGF23的mRNA表达变化,结果与空白对照组比较。结果 体外培养成骨细胞生长良好,成骨细胞FGF23的mRNA水平在汇合时显著上调,为半汇合阶段的7.5倍（P<0.001）,于基质堆积和矿化时期回落至半汇合水平,提示成骨细胞FGF23的表达呈阶段特异性。10^-8 mol/L 1,25(OH)₂D₃刺激后,FGF23的mRNA表达水平明显上调,约为对照组的16倍（P<0.001）;而CaCl₂、βGP、PTH刺激后,FGF23的mRNA表达水平与对照组相比,差异无统计学意义。结论 FGF23的表达与成骨细胞的分化状态有关,1,25(OH)₂D₃能强烈刺激体外培养成骨细胞FGF23的表达。

Fibroblast growth factor 23 expression in rat calvarial osteoblastic cells and its responses to hormones in vitro

Objective To investigate the expression patterns of fibroblast growth factor 23(FGF23) in osteoblast and its responses to calcium,phosphate,exogenous PTH and 1,25(OH)2D3. Methods The primary rat calvarial osteoblasts were cultured in MEM medium which containing 10% FBS,then were harvested when cells were in half-confluence,confluence,osteoid deposition and osteoid mineralization stages respectively.The procedure was monitored under microscopy.Total RNA was extracted from cells according to the Trizol proced...