orexin-1受体抑制剂通过Wnt通路促进骨髓间充质干细胞的成骨分化

In vitro study on the effect of Wnt signaling pathway on orexin-1 receptor inhibitor induced osteogenic differentiation of rat bone marrow mesenchymal stem cells

目的研究Wnt/β-catenin信号通路对orexin-1受体抑制剂介导大鼠骨髓间充质干细胞(BMSCs)成骨分化的影响。方法(1)分离提取SD大鼠股骨、胫骨的BMSCs,用流式细胞仪进行细胞表型鉴定;(2)实验分组:对照组、10-5mol/L、10-6mol/L质量浓度的orexin-1受体抑制剂溶液,于成骨诱导第5天免疫印迹法和实时荧光定量PCR法观察Wnt/β-catenin信号通路关键靶点蛋白Dickkopf-1、Gsk3β、β-catenin以及基因Gsk3β、β-catenin mRNA表达,以观察Wnt信号通路对orexin-1受体抑制剂诱导大鼠骨髓间充质干细胞成骨分化的影响。结果 10^(-5)mol/L、10^(-6)mol/L的orexin-1受体抑制剂处理BMSCs5天后,Dickkopf-1、β-catenin和GSK3β蛋白表达升高,β-catenin和GSK3βmRNA表达升高。结论 orexin-1受体抑制剂促进大鼠骨髓基质干细胞向成骨细胞方向分化的作用可能与调控Wnt/β-catenin信号通路有关。

Objective To observe the effect of Wnt/intracellular beta chain protein（ β-catenin） signaling pathway on orexin-1receptor inhibitor induced osteogenic differentiation of bone marrowmesenchymal stem cells（ BMSCs） in rats. Methods（ 1）Isolated the bone marrowmesenchymal stem cells from one-month SD rats by whole bone marrowculture method and identified the cell surface antigen by flowcytometry.（ 2） Experimental groups： control group,and 10-5mol/L or 10-6mol/L orexin-1 receptor inhibitor solution groups. At day 5 the mRNA expression of Gsk3 beta and beta-catenin were detected by real-time quantitative PCR.The protein expression of Dickkopf1,Gsk3 beta and beta-catenin were detected by Western blot. Results Orexin-1 receptor inhibitor up-regulated Gsk3 beta and beta-catenin mRNA expression at concentrations of 10-6mol/L and 10-5mol/L for 5 days.Orexin-1 receptor inhibitor up-regulated Dickkopf1,Gsk3 beta and beta-catenin protein expression at concentrations of 10-6mol/L and 10-5mol/L for 5 days（ P 〈 0. 05）. Conclusion The effect of orexin-1 receptor inhibitor on osteogenic differentiation of MSCs maybe closely related to the regulation of Wnt/catenin signaling pathway.