活血接骨方对骨髓间充质干细胞成骨分化中Wnt信号通路及Sclerostin基因的影响

Effects of Huoxue Jiegu Formula on Wnt Signaling Pathway and Sclerostin Gene in Osteogenic Differentiation of Bone Marrow Mesenchymal Stem CellsAuthor

目的探究活血接骨方对骨髓间充质干细胞成骨分化中Wnt信号通路及Sclerostin基因的影响。方法使用"续骨活血汤"含药血清和(或)BMP-4干预BMSCs并分为4组:对照组、中药组、BMP-4组和中药+BMP-4组。通过检测线粒体脱氢酶活性(CCK-8)及细胞DNA含量反映BMSCs细胞活性。采用ALP染色和茜素红染色检测成骨诱导28天后各组细胞ALP活性和矿化情况;采用荧光定量PCR和Western Blot法检测各组细胞中成骨相关基因(Col1A2、OC、OPN)和Sclerostin基因表达情况。使用(低浓度、中浓度、高浓度)活血接骨方含药血清体外干预BMSCs 7天后检测Sclerostin基因及Wnt信号通路相关分子(Wnt5a、GSK-3β)的表达情况。结果与对照组相比,其他3组BMSCs出现明显矿化,且BMP-4组矿化程度高于中药组,而中药+BMP-4组矿化程度最高(P<0.05);成骨相关因子Col1A2、OC、OPN也呈现相同趋势的表达上调(P<0.05),但Sclerostin基因呈现明显表达下调(P<0.05)。与空白对照组相比,各浓度含药血清组BMSCs中Wnt5a的表达呈现浓度依赖性上调(P<0.05),而Sclerostin、GSK-3β和P-GSK-3β的表达则呈现浓度依赖性下调(P<0.05)。结论活血接骨方具有促进BMSCs体外成骨分化作用,其机制可能与抑制Sclerostin基因表达,激活Wnt信号通路相关。

Objective To explore the effects of the Huoxue Jiegu Formula on the Wnt signaling pathway and Sclerostin gene in the osteogenic differentiation of bone marrow derived mesenchymal stem cells.Methods BMSCs were treated with Huoxue Jiegu Formula containing serum and/or BMP-4 and divided into 4 groups:control group,Chinese medicine group,BMP-4 group and Chinese medicine+BMP-4 group.The cellular activity of BMSCs was measured by mitochondrial dehydrogenase activity(CCK-8)and cellular DNA content.ALP activity and mineralization of cells in each group after 28 days of osteogenesis induction were detected by ALP staining and alizarin red staining;osteogenesisrelated genes(Col1 A2,OC,OPN)and Sclerostin gene expression in each group of cells were detected by fluorescence quantitative PCR and Western Blot.The expression of Sclerostin gene and Wnt signaling pathway-related molecules(Wnt5 a,GSK-3β)were detected in BMSCs after 7 days of in vitro intervention with(low,medium and high concentrations of)the blood-raising and bone-setting formula containing serum.Results Compared with the control group,after 28 days of in vitro intervention,there was obvious mineralization of BMSCs in the Chinese medicine group and the BMP-4 group,and the BMP-4 group was stronger than the Chinese medicine group,while the Chinese medicine+BMP-4 group had the highest degree of cell mineralization(P<0.05).Osteogenic factors such as Col1 A2,OC and OPN were also up-regulated(P<0.05),but Sclerostin was significantly down-regulated(P<0.05).Compared with the blank control group,the mRNA and protein levels of Wnt5 a in BMSCs with different concentrations of drug containing serum were up-regulated in a concentration-dependent manner(P<0.05),while the mRNA and protein levels of Sclerostin,GSK-3βand P-GSK-3βwere down-regulated in a concentration-dependent manner(P<0.05).Conclusion The Huoxue Jiegu Formula has the effect of promoting osteogenic differentiation of BMSCs in vitro,and the mechanism may be related to the inhibition of Sclerostin gene expression and activation of Wnt signaling pathway.