黄精多糖调控骨髓间充质干细胞向成骨细胞分化

Polygonatum sibiricum polysaccharide regulates osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells

背景:骨髓间充质干细胞具有多向分化潜能。黄精多糖可能通过Wnt/β-catenin信号通路促进小鼠骨髓间充质干细胞的成骨分化。目的:探讨黄精多糖对小鼠骨髓间充质干细胞向成骨细胞分化过程中Wnt/β-catenin信号通路的影响。方法:培养小鼠骨髓间充质干细胞,在传统成骨诱导培养基诱导下给予5,10,25,50 mg/L黄精多糖进行干预,不加黄精多糖作为对照组,显微镜下观察细胞形态变化。PNPP法检测碱性磷酸酶活性;观察细胞发生矿化并进行茜素红染色,记录矿化结节数和面积分数;q RT-PCR检测成骨相关基因碱性磷酸酶、Runx2、骨钙素m RNA表达;采用q RT-PCR和Western Blot分别检测β-catenin基因和蛋白表达水平;采用双荧光素酶报告基因检测系统检测下游β-catenin/TCF的转录活性。结果与结论:(1)与对照组相比,不同质量浓度的黄精多糖以剂量依赖性的方式提高碱性磷酸酶活性(P<0.05),显著增强细胞的矿化能力,明显提高碱性磷酸酶、Runx2、骨钙素基因的表达(P<0.05);(2)诱导后β-catenin m RNA在第3天表达最高,黄精多糖在促进骨髓间充质干细胞向成骨细胞分化的过程中还能显著上调β-catenin的表达(P<0.05),促使含有TCF结合位点的荧光素酶报告基因(TOPFlash)的高表达(P<0.05);(3)说明黄精多糖通过激活Wnt/β-catenin信号通路促进小鼠骨髓间充质干细胞向成骨细胞分化。

BACKGROUND： Bone marrow mesenchymal stem cells（BMSCs） have the ability of multi-directional differentiation. Polygonatum sibiricum polysaccharide can promote osteogenetic differentiation of mouse BMSCs by activating Wnt/β-catenin signaling pathway, which is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE： To investigate the effects of Polygonatum sibiricum polysaccharide on Wnt/β-catenin signaling pathway in the osteogenic differentiation of mouse BMSCs. METHODS： The mouse BMSCs were cultured and induced in osteoblast medium containing final concentrations（5, 10, 25, 50mg/L） of Polygonatum sibiricum polysaccharide. The mouse BMSCs treated without Polygonatum sibiricum polysaccharide was set as the negative control group. The morphological changes of cells were observed under an inverted microscope. Alkaline phosphatase（ALP） activity assay was performed by PNPP method. The mineralization nodules were observed and stained with alizarin red S and the number and area fraction were recorded under an inverted microscope. The m RNA expressions of osteogenesis-related genes ALP, Runx2, and osteocalcin were evaluated by quantitative real-time PCR（q RT-PCR）. q RT-PCR and western blot were used to determine the expression level of β-catenin. The downstream β-catenin/TCF transcriptional activity was evaluated with the Dual-Luciferase Reporter Assay System. RESULTS AND CONCLUSION： Compared with the control group, polygonatum sibiricum polysaccharide significantly enhanced the alkaline phosphatase activity, the mineralization ability of cells, and the m RNA expression of ALP, Runx2 and osteocalcin in the differentiated BMSCs in a dose dependent manner（P〈0.05）. After induction, the m RNA expression of β-catenin was the highest on the 3rd day. Polygonatum sibiricum polysaccharide significantly increased the expression of β-catenin（P〈0.05） in the process of promoting the differentiation of BMSCs into osteoblasts, and also promoted the high-level expression of luciferase reporter gene（TOPFlash） which contains wild type TCF binding sites（P〈0.05）. These results demonstrate that Polygonatum sibiricum polysaccharide can promote the osteoblast differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway.