神经营养因子-3通过Wnt通路促进人骨髓间充质干细胞生长和成骨分化的研究

Neurotrophin-3 promotes growth and osteogenic differentiation of human bone marrow mesenchymal stem cells through Wnt pathway

目的阐明神经营养因子-3(NT-3)促进人骨髓间充质干细胞分化为成骨细胞的能力,并分析Wnt通路的作用机制。方法正常培养的人骨髓间充质干细胞为对照组;NT-3刺激的人骨髓间充质干细胞为NT-3组;加入ICG-001作用30 min后,用NT-3刺激者为Wnt抑制剂组,每组进行成骨诱导实验。利用噻唑蓝细胞增殖、酶联免疫吸附试验、Western blot检测、茜素红染色等实验分别检测各组人骨髓间充质干细胞增殖、细胞凋亡、碱性磷酸酶、骨形态发生蛋白-1(BMP-1)等蛋白表达及钙结节形成能力。结果与对照组比较,NT-3组间充质干细胞(MSC)增殖吸光度值增高(P<0.01);NT-3组碱性磷酸酶活性、BMP-1等蛋白表达或茜素红染色效果均高于对照组(P<0.01);与NT-3组比较,Wnt抑制剂组MSC增殖吸光度值降低(P<0.01);而Wnt抑制剂组的碱性磷酸酶活性、BMP-1等蛋白表达均低于NT-3组(P<0.01)。结论 NT-3通过Wnt通路,促进人骨髓间充质干细胞的增殖和成骨分化。

Objective To clarify the ability of neurotrophin-3 （NT-3） in promoting the differentiation of human bone marrow mesenchymal stem cells （hMSCs） into osteoblasts, and analyze the mechanism of Wnt pathway. Methods Normal cultured hMSCs were into control group, hMSCs stimulated by NT-3 were into NT-3 group, hMSCs added with ICG-001 acting for 30 min before stimulation of NT-3 were taken as Writ inhibitor group. The experiment of ostcogenic induction was performed in each group. MTT, ELISA, Western blot and alizarin red staining were used to detect hMSC proliferation, apoptosis, alkaline phosphatase activity （ALP）, bone morphogenetic protein-I （BMP-1） and other proteins, and ability of calcium nodule formation res- pectively in each group. Results Compared with the control group, the OD value of hMSC proliferation in the NT-3 group significantly increased （P〈0.01）; the expressions of ALP, BMP-1 and other proteins, and the effect of alizarin red staining of the NT-3 group were significantly higher than those in the control group （P 〈 0.01）. Compared with the NT-3 group, the OD value of hMSC proliferation in the Wnt inhibitor group signifi- cantly decreased （P〈 0.01）; the expressions of ALP, BMP-1 and other proteins of the Wnt inhibitor group were lower than those in the NT-3 group （P〈 0.01）. Conclusions NT-3 promotes the proliferation and osteo- genic differentiation of hMSCs through Wnt pathway