骨碎补含药血清经wnt/β-catenin信号通路对大鼠骨髓间充质干细胞成骨分化的影响

Effect of Rhizoma drynariae drug-containing serum on osteogenic differentiation of bone marrow mesenchymal stem cells by wnt/beta-catenin signaling pathway

目的研究骨碎补含药血清经wnt/β-catenin信号通路对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的作用,探讨其可能的作用机制。方法用不同浓度含药血清干预BMSCs,CCK-8法分别检测血清干预BMSCs 3、5、7、9 d BMSCs的增殖能力,连续诱导7、10、14 d并进行ALP活性检测,诱导21 d后茜素红染色评价BMSCs的矿化能力,RT-PCR法检测β-catenin、LRP5、GSK-3β、RUNX-2及Osteriex mRNA表达;ELISA法检测细胞内β-catenin、LRP5蛋白表达量。结果骨碎补含药血清具有明显的促进BMSCs增殖的作用,在一定的时间段提高了ALP活性,促进钙化结节形成;上调wnt/β-catenin信号通路β-catenin、LRP5、RUNX-2及Osteriex mRNA表达;下调GSK-3βmRNA表达,上调β-catenin、LRP5蛋白表达。结论骨碎补含药血清可以促进BMSCs的增殖及成骨分化,其机制与激活wnt/β-catenin信号通路,上调β-catenin、LRP5、RUNX-2及Osteriex mRNA表达,下调GSK-3βmRNA表达及上调β-catenin、LRP5蛋白表达密切相关。

Aim To investigate the drug-containing serum of Rhizoma drynariae on osteogenesis differentiation of bone marrow mesenchymal stem,and discuss the possible mechanism. Methods BMSCs were cultured in media with different concentrations of medicine containing serum. BMSCs proliferation ability was detected in 3,5,7,9 days by CCK-8. ALP activity was detected after 7,10,14 days' induction. After 3 weeks culturing,alizarin red staining was performed to observe the formation of calcium nodules. The expression of β-catenin,LRP5,RUNX-2 and Osteriex mRNA were detected using RT-PCR. The protein expression of β-catenin,LRP5 was detected using Elisa method. Results Rhizoma drynariae drug-containing serum could obviously promote the proliferation of BMSCs and calcified nodule formation. Besides,the ALP activity wasimproved in a certain period of time. The expression ofβ-catenin,LRP5,GSK-3β,RUNX-2 and Osteriex mRNA were significantly up-regulated,and the protein expression of β-catenin,LRP5 was up-regulated too. The expression of GSK-3β was down-trgulated. Conclusions Rhizoma drynariae drug-containing serum promotes mineralization and osteogenic differentiationof BMSCs,and the mechanism is closely related with activating WNT/beta-catenin signaling pathway,raisingthe beta-catenin,LRP5,RUNX-2,and Osteriex mRNA expression, beta-catenin, LRP5 protein expression,and down-regulation of GSK-3β mRNA expression.