补肾填精中药血清对去势小鼠骨髓干细胞Wnt/β-catenin成骨分化信号通路的影响

Effect of invigorating kidney and nourishing essence Chinese medicine containing serum on Wnt/β-catenin osteogenic differentiation signal pathway in BMSCs in ovariectomized mice

目的探讨补肾填精中药血清对小鼠骨髓干细胞增殖、分化、矿化功能的影响,并检测Wnt/β-catenin信号通路相关蛋白的表达,深化"肾主骨、生髓"理论的科学内涵。方法以不同浓度含药血清培养去势小鼠骨髓干细胞,MTT观察细胞增殖,连续培养1周后,检测成骨基因的表达并进行碱性磷酸酶染色,培养28天后茜素红染色观察钙结节的形成,Elisa及Western-blot检测Wnt/β-catenin通路主要信号蛋白β-连环蛋白(β-catenin)、低密度脂蛋白受体相关蛋白5(LRP-5)、T细胞因子(TCF)的表达水平。结果补肾填精含药血清促进了去势小鼠BMSCs的增殖、成骨基因的表达和钙结节的形成,β-catenin、LRP-5、TCF显著上调。结论补肾填精中药血清促进了小鼠BMSCs的成骨分化与矿化能力,这种促进作用与Wnt/β-catenin信号通路存在密切关系。

Objective To investigate the effect of invigorating kidney and nourishing essence Chinese medicine containing serum on the proliferation, osteogenie differentiation, and mineralization of mouse bone marrow mesenchymal stem cells （BMSCs） , to detect the expression of relevant proteins of Wnt/β-catenin signal pathway, and to deepen the scientific connotation of the theory that ＂the kidney governs the bone and produces the marrow＂. Methods BMSCs isolated from ovariectomized mice were cultured in media with different concentrations of medicine containing serum. Cell proliferation was determined using MTT assay. After 1-week culturing, osteogenic gene expression was detected. And alkaline phosphatase （ALP） staining was performed. Alizarin red staining was performed to observe the formation of calcium nodules after 28-day culturing. The expression of β-catenin, low-density lipoprotein receptor-related protein 5 （LRP-5） , and T cell factor （TCF） , the major signaling proteins of Wnt/β-catenin pathway, were detected using Elisa method and Western blotting. Results Invigorating kidney and nourishing essence medicine containing serum could promote the proliferation of BMSCs, the expression of osteogenie gene, and the formation of calcium nodules. The expression of β-catenin, LRP-5, and TCF was significantly up-regulated. Conclusion Invigorating kidney and nourishing essence Chinese medicine containing serum promotes osteogenic differentiation and mineralization of BMSCs in ovarieetomized mice. And this promoting function is closely correlated with Wnt/β-eatenin signal pathway.