醛固酮对成骨细胞增殖分化及成骨相关基因表达的影响

目的 探讨外源性醛固酮对大鼠原代培养的成骨细胞增殖、碱性磷酸酶活性及成骨相关基因表达的影响及其机制.方法采用酶消化法体外培养大鼠成骨细胞,CCK-8试剂盒和AKP试剂盒分别检测成骨细胞的增殖和碱性磷酸酶活性情况,半定量RT-PCR和Western blotting检测成骨细胞骨形成活性相关标志物和上皮钠离子通道(α-ENaC)基因的mRNA和蛋白表达.结果与对照组相比,醛固酮浓度为1×10-2~1μmol/L范围内可以促进成骨细胞增殖,浓度为1×10-3μmol/L和10μmol/L时对成骨细胞增殖无显著影响;但醛固酮在1×10-3~10μmol/L浓度范围内对成骨细胞碱性磷酸酶活性均无明显影响.与对照组相比,醛固酮浓度为1×10-2~1μmol/L时均能上调成骨细胞中成骨细胞骨形成活性相关标志物I型胶原蛋白(Coll-Ia)、碱性磷酸酶(ALP)、骨桥蛋白(OPN)和α-ENaC基因的mRNA和蛋白的表达水平(P<0.05).结论醛固酮(1×10-2~1μmol/L)能明显促进成骨细胞增殖并上调成骨细胞骨形成活性相关标志物的表达,且同时上调ENaC基因的表达,提示ENaC可能是醛固酮调节成骨细胞功能的作用靶点之一.

Effects of aldosterone on osteoblast proliferation,differentiation and osteogenic gene expressions in vitro

Objective To study the effect of aldosterone on cell proliferation, alkaline phosphatase (AKP) activity and osteogenic gene expression in rat osteoblasts and explore the mechanisms. Methods Osteoblasts isolated from the skull of neonatal SD rats by enzyme digestion were cultured and treated with different concentrations of aldosterone. The cell proliferation and AKP activity were evaluated using CCK-8 assay kit and AKP assay kit, respectively. The effects of aldosterone on mRNA and protein expressions of the osteogenic genes and epithelial sodium channel (ENaC) gene were investigated using semi-quantitative PCR and Western blotting. Results Compared with the control cells, the cells treated with 0.01-1.0 μmol/L aldosterone showed obviously enhanced proliferation while lower (1 × 10-3 μmol/L) or higher (10 μmol/L) concentrations of aldosterone did not significantly affect the cell proliferation. Aldosterone within the concentration range of 1× 10-3 to 10 μmol/L did not cause significant changes in AKP activity in the osteoblasts. Treatment with 0.01 to 1.0 μmol/L aldosterone significantly upregulated the expressions of the osteogenic genes andα-ENaC gene at both the mRNA and protein levels. Conclusion Aldosterone within the concentration range of 0.01-1.0 μmol/L stimulates the proliferation and osteogenic gene expressions and enhances α-ENaC gene expression in rat osteoblasts in vitro, suggesting the possibility that ENaC participates in aldosterone-mediated regulation of osteoblast functions.