人重组骨形成蛋白2作用下人牙周膜细胞Osterix的表达变化

Expression of Osterix mRNA and protein induced by recombinant human bone morphogenetic protein-2 in human periodontal ligament cells

目的观察人重组骨形成蛋白2（rhBMP-2）作用下，人牙周膜细胞内Osterix（Osx）mRNA和蛋白的表达变化，初步探讨牙周膜细胞成骨分化过程中BMP-2与Osx的信号传递途径。方法组织块法培养人牙周膜细胞至3代。用含rhBMP-2的培养液，按照不同浓度、不同时间分组进行培养，后采用实时定量反转录（Real—timeRT）-PCR和Western印迹法分别检测各组细胞OsxmRNA和蛋白表达变化。选用SB203580抑制细胞p38磷酸化，检测rhBMP．2诱导过程中磷酸化p38（P．p38）蛋白、OsxmRNA表达及细胞矿化反应变化。结果在rhBMP-2持续作用下，人牙周膜细胞内OsxmRNA表达明显升高并具有时效性；Osx蛋白表达从无到有，并逐渐增强。当rhBMP~浓度≤200斗g／L时，OsxmRNA和蛋白表达呈现为剂量依赖性增强；当rhBMP-2〉200μg／L时，Osx表达趋于平缓。在p38抑制剂SB203580干扰下，rhBMP-对p-p38及Osx的诱导增强作用被显著抑制（OsxmRNA表达：0．378±0．034比0．134±0．027，Osx蛋白表达：0．353±0．024比0．155±0．031，均P〈0．01），矿化结节形成亦相对较少且滞后。结论BMP_2对Osx具有显著正向调控作用，p38途径是此信号传递过程中的一个重要环节，BMP-2／p38／Osx通路可能是牙周膜细胞成骨分化过程中的一条重要信号通路。

Objective To detect the changes of Osterix （Osx） mRNA and protein expression in human periodontal ligament cells （HPDLCs） induced by recombinant human bone morphogenetic protein-2 （rhBMP-2）, and examine the role of BMP-2 and Osx during the osteogenic differentiation of HPDLCs. Methods HPDLCs were isolated and cultured in vitro with explant method. Ceils at passage 3 were cultured in DMEM with rhBMP-2 at different concentrations （50, 100, 150,200,250,300,400, and 600 I.Lg/L） for different times （2, 3, 5, 7, 10, 14 and 21 days）. Then the expressions of Osx mRNA and protein were measured by real-time reverse transcription （RT） -PCR and Western blotting respectively. Cells were treated with 10 μmol/L SB203580 （p38 inhibitor） to inhibit p38 phosphorylation induced by rhBMP-2. The mineralization nodules formation and the expressions of phosphorylated p38 and Osx mRNA were detected respectively. Results During the culture of rhBMP-2, the expression of Osx mRNA significantly increased. Initially Osx protein had a low expression and then increased in a time-dependent manner followed by the production of bone-like nodules in HPDLCs. Under the effect of SB203580, the up-regulation of phosphorylated p38 expression induced by rhBMP-2 was significantly inhibited as well as the expression of Osx （ Osx mRNA expression： 0. 378 ± 0. 034 vs 0. 134 ± 0. 027, Osx protein expression ： 0. 353 ＋ 0. 024 vs 0. 155 ＋ 0. 031, both P 〈 0. 01 ）. Meanwhile the mineralization nodules formed by HPDLCs induced by rhBMP-2 were fewer and delayed. Conclusions BMP-2 has a significant positive regulatory role on the expression of Osx in HPDLCs. And p38 pathway is an important link of this regulatory process. Thus, as an important signaling pathway in osteogenic differentiation of HPDLCs, BMP-2/p38/Osx may be involved in periodontal tissue remodeling.