皮质酮浓度对SD大鼠成骨细胞成骨基因表达的影响

Effect of corticosterone concentration on osteoblastic gene expression of SD rat osteoblasts

目的探讨皮质酮对离体SD大鼠成骨细胞功能的影响。方法采用多次胶原酶组织消化法获得新生SD大鼠颅骨中的成骨细胞作为研究对象,用倒置显微镜及碱性磷酸酶、钙结节染色观察成骨细胞形态,CCK-8法检测皮质酮对成骨细胞增殖的影响,根据CCK-8结果将SD大鼠成骨细胞分成四组,分别用含不同浓度的皮质酮(0μmol/L、0.1μmol/L、1.0μmol/L、10.0μmol/L)的DMEM(H)培养基培养,作用24 h后,测定成骨细胞内碱性磷酸酶含量,采用RT-PCR检测成骨细胞内COL1A、OCN、ALP基因表达,采用Western blot检测成骨细胞内COL1A、OCN、RUNX-2蛋白的表达。结果 CCK-8结果显示皮质酮能抑制成骨细胞的增殖,皮质酮能抑制成骨细胞内碱性磷酸酶的生成,与浓度明显相关;RT-PCR与Western blot检测可见COL1A、OCN、ALP基因表达及COL1A、OCN、RUNX-2蛋白的表达下降。结论皮质酮在超生理剂量的浓度下能抑制成骨细胞增殖及细胞内碱性磷酸酶的合成,减少COL1A、OCN、ALP基因及COL1A、OCN、RUNX-2蛋白的表达。

Objective To investigate the effect of different corticosterone concentration on the gene expres-sion of SD rat osteoblasts in vitro. Methods Newborn SD rat osteoblasts in the skull were obtained by many times of collagenase digestion as the object of study. The osteoblastic morphology was observed using inverted microscope, al-kaline phosphatase and calcium nodes dyeing. And the effect of corticosterone concentration on osteoblastic prolifera-tion was detected by CCK-8. According to the scores of CCK-8, the SD rat osteoblasts were divided into four groups with different corticosterone concentrations of DMEM （H） culture （0μmol/L, 0.1μmol/L, 1.0μmol/L, 10.0μmol/L）. After 24 hours of DMEM （H） culture, content of osteoblastic alkaline phosphatase were measured, gene expressions of osteoblastic COL1A, OCN, ALP were detected by RT-PCR, and the protein expression of osteoblastic COL1A, OCN, RUNX-2 were measured by western blot. Results The scores of CCK-8 showed that corticosterone can re-strain the proliferation of SD rat osteoblasts and the formation of osteoblastic alkaline phosphatase, which were signifi-cantly related to concentration. And the results of RT-PCR and western blot both showed decreased expression of COL1A, OCN, ALP gene and COL1A, OCN, RUNX-2 protein. Conclusion Corticosterone beyond physical dosage can inhibit the proliferation of osteoblasts and synthesis of intracellular alkaline phosphatase, and reduce the expres-sion of COL1A, OCN, ALP gene and COL1A, OCN, RUNX-2 protein.