不同浓度地塞米松体外对人成骨细胞和破骨细胞分化的影响

Effect of dexamethasone on differentiation of human osteoblast and osteoclast in vitro

目的:探讨地塞米松(DEX)体外对人成骨细胞(OBs)和破骨细胞(OCs)分化的影响。方法:以DEX为主要成分的诱导剂体外刺激正常人骨髓间充质干细胞(MSCs),茜素红染色及碱性磷酸酶(ALP)定量法检测OBs间钙结节形成及OBs中ALP水平;改变DEX浓度后,再检测ALP水平以及用Western blotting检测OBs中磷酸化GSK-3β蛋白的表达;外源性核因子κB受体活化因子配基(RANKL)刺激人骨髓单个核细胞(BMMs)14 d后,用抗酒石酸酸性磷酸酶染色鉴定OCs形成情况、Western blotting检测BMMs中RANKL下游的信号途径;不同浓度DEX刺激MSCs 7 d后,用ELISA和Western blotting检测培养液中可溶性RANKL(sRANKL)水平及MSCs中RANKL蛋白表达。结果:含0.1μmol/L DEX的诱导剂刺激MSCs后可有钙结节形成,在DEX 0.001-0.1μmol/L浓度范围内,OBs中ALP水平逐渐增高、磷酸化GSK-3β蛋白表达逐渐增强,当DEX浓度进一步增加后(0.1-10μmol/L),ALP水平和磷酸化GSK-3β蛋白表达反而下降;外源性RANKL可以诱导BMMs分化为OCs,且BMMs中磷酸化IκBα、SPAK/JNK、p38 MAPK、p44/42 MAPK表达增强;在0.001-10μmol/L浓度范围内,DEX刺激MSCs后,培养液中sRANKL水平和MSCs中RANKL蛋白表达逐渐增高,且sRANKL水平和DEX浓度呈正相关(r=0.821,P<0.05)。结论:在低浓度(0.001-0.1μmol/L)范围内,DEX既促进OBs分化,又可促进OCs分化。在高浓度(0.1-10μmol/L)范围内,仍能促进OCs分化,但失去了其对OBs的分化作用。

AIM： To investigate the effect of dexamethasone（DEX） on differentiation of human osteoblasts（OBs） and osteoclasts（OCs） in vitro.METHODS： The normal human bone marrow mesenchymal stem cells（MSCs） were stimulated with the inducer mainly containing DEX in vitro.Alizarin red staining and alkaline phosphatase（ALP） kit were used to identify the formation of calcium nodules among OBs and ALP levels in OBs,respectively.The levels of ALP were quantified again after the concentration of DEX was changed,and the expression of phosphorylated GSK-3β in OBs was detected by Western blotting.Human bone marrow mononuclear cells（BMMs） were induced by exogenous receptor activator of nuclear factor kappa B ligand（RANKL） for 14 days.Tartrate-resistant acidic phosphatase staining was used to identity the formation of OCs,and the downstream signaling pathway of RANKL was detected by Western blotting.The le-vel of soluble RANKL（sRANKL） in the supernatants and the expression of RANKL in MSCs were measured by ELISA and Western blotting,respectively,when MSCs were stimulated with different concentrations of DEX for 7 days.RESULTS： Calcium nodules were formed when MSCs were stimulated with DEX at concentration of 0.1 μmol/L.The level of ALP and the protein expression of phospho-GSK-3β in OBs gradually increased by treating the cells with DEX at the concentrations within the range of 0.001-0.1 μmol/L.However,the level of ALP and the protein expression of phospho-GSK-3β were decreased as the concentrations of DEX further increased to the range of 0.1-10 μmol/L.BMMs were differeniated into OCs by exogenous RANKL,and the phosphorylations of IκBα,SPAK/JNK,p38 MAPK and p44/42 MAPK in BMMs increased.When MSCs were stimulated with DEX at the concentrations within 0.001-10 μmol/L,the level of sRANKL in the supernatants and the protein expression of RANKL in MSCs gradually increased,and the level of sRANKL was positively correlated with the concentration of DEX（r=0.821,P0.05）.CONCLUSION： DEX at low concentrations promotes the differentiation of OBs and OCs.DEX at high concentrations only promotes the differentiation of OCs,not that of OBs.