补肾活血胶囊含药血清基于Wnt/β-Catenin信号通路促进骨髓间充质干细胞成骨分化研究

Study on serum containing Bushen Huoxue Capsules promoting bone marrow mesenchymal stem cells osteogenic differentiation based on Wnt/β-Catenin signaling pathway

目的:研究补肾活血胶囊含药血清基于Wnt/β-Catenin信号通路促进骨髓间充质干细胞成骨分化机制。方法:全骨髓培养法获取SD大鼠骨髓间充质干细胞(rBMSCs);细胞形态学及表面抗原鉴定rBMSCs;采用CCK-8法、碱性磷酸酶(ALP)法和茜素红染色检测不同剂量补肾活血胶囊含药血清及加入Wnt信号通路特异性阻滞剂DKK-1干预的rBMSCs增殖及ALP、成骨分化活性;RT-PCR及Western Blot测定rBMSCs Wnt3a、β-Catenin、c-myc、CyclinD1、Runx2蛋白及mRNA表达水平。结果:全骨髓培养法获取P3代rBMSCs纯度高,各剂量实验组含药血清可促进rBMSCs增殖、ALP活性及成骨分化,各剂量抑制剂组较同剂量实验组显著抑制rBMSCs增殖、ALP活性及成骨分化(P<0.05)。各剂量实验组含药血清可促进Wnt3a、β-Catenin、c-myc、CyclinD1、Runx2蛋白及mRNA表达,各剂量抑制剂组较同剂量实验组显著抑制Wnt3a、β-Catenin、c-myc、CyclinD1、Runx2蛋白及mRNA表达(P<0.05)。结论:补肾活血胶囊可以促进骨髓间充质干细胞增殖、成骨分化,其机制可能与调节Wn t/β-Catenin信号通路有关。

Objective:To study mechanism of serum containing Bushen Huoxue Capsulespromoting rBMSCs osteogenic differentiation based on Wnt/β-Catenin signaling pathway.Methods:The BMSCs of SD rats were obtained by whole bone marrow culture method.The rBMSCs were identified by Cell morphology and surface antigen identification.The rBMSCs proliferation and ALP,osteogenic differentiation activity of Bushen Huoxue Capsules containing serum and adding Wnt signal pathway specific blocker were detected by CCK-8 method,alkaline phosphatase(ALP)method and alizarin red staining.The protein and mRNA expression of Wnt3a,β-Catenin,c-myc,CyclinD1,Runx2 were determined by RT-PCR and Western Blot.Results:The P3 generation rBMSCs obtained by whole bone marrow culture method were high purity.The serum of Bushen Huoxue Capsules could promote rBMSCs proliferation,ALP activity and osteogenic differentiation.The DKK-1 inhibitor groups inhibited rBMSCs proliferation,ALP activity and osteogenic differentiation(P<0.05).The serum of Bushen Huoxue Capsules can promote the protein and mRNA expression of Wnt3a,β-Catenin,c-myc,CyclinD1,Runx2.The protein and mRNA expression of Wnt3a,β-Catenin,c-myc,CyclinD1,Runx2 were inhibited by each dose inhibitor group(P<0.05).Conclusion:Bushen Huoxue Capsulescan promote the proliferation and osteogenic differentiation of BMSCs,and its mechanism may be related to the regulation of Wnt/β-Catenin signaling pathway.