刘氏正骨方对BMSCs成骨分化中Wnt信号通路及Sclerostin基因的影响

Effect of Liu′s Zhenggu-Fang on Wnt Signaling Pathway in Osteogenic Differentiationof Bone Marrow Mesenchymal Stem Cells

目的探究刘氏正骨方对骨髓间充质干细胞成骨分化中Wnt信号通路的影响。方法使用刘氏正骨方含药血清或BMP-2对骨髓间充质干细胞(BMSCs)进行28 d成骨诱导后,检测BMSCs的细胞活性、ALP活性及细胞矿化情况;采用RT-PCR和Western blotting法检测细胞中Col 1A2、OCN、OPN和Sclerostin基因表达情况。使用含2.5%、5%、10%中药血清对BMSCs进行14 d体外干预,检测Wnt信号通路关键因子LRP5、Sclerostin及成骨关键转录因子Runx2的表达情况。结果1)中药组和BMP-2组中BMSCs中ALP活性及细胞矿化现象明显强于对照组,且中药组明显高于BMP-2组(P<0.05);Col1A2、OCN、OPN的表达也呈现相同趋势改变(P<0.05),但Sclerostin基因呈现明显表达下调(P<0.05)。2)高浓度中药血清可上调LRP5和Runx2表达(P<0.05),下调Sclerostin基因表达(P<0.05)。结论刘氏正骨方可以促进BMSCs体外成骨分化,其作用机制可能与下调Sclerostin基因表达、激活Wnt信号通路有关。

Objective:To explore the effect of Liu’s Zhenggu-Fang on Wnt signaling pathway in the osteogenic differentiation of bone marrow mesenchymal stem cells.Methods:After 28 days of osteogenic induction of BMSCs with Liu’s Zhenggu-Fang-containing serum or BMP-2,the cell activity,ALP activity and cell mineralization of BMSCs were detected;RT-PCR and Western blotting were used to detect Col1 A2 in cells OCN,OPN and Sclerostin gene expression.The serum containing 5%,10%,and 20%Chinese medicine was used to intervene BMSCs in vitro for 14 days to detect the expression of key factors of Wnt signaling pathway LRP5,β-catenin and key osteogenic transcription factor Runx2.Results:1)ALP activity and cell mineralization in BMSCs in the traditional Chinese medicine group and BMP-2 group were significantly stronger than those in the control group,and the BMP-2 group was significantly higher than the traditional Chinese medicine group(P<0.05);the expression of Col1 A2,OCN and OPN were also It showed the same trend change(P<0.05),but the expression of Sclerostin gene was significantly down-regulated(P<0.05).2)High-concentration traditional Chinese medicine serum can up-regulate the expression of LRP5,β-catenin and Runx2(P<0.05),and down-regulate the expression of Sclerostin gene(P<0.05).Conclusion:Liu’s Zhenggu-Fang can promote osteogenic differentiation of BMSCs in vitro,and its mechanism may be related to down-regulation of Sclerostin gene expression and activation of Wnt signaling pathway.