目的 探讨Toll样受体(TLR)4激活后调控Wnt/β-catenin信号通路促进骨髓间充质干细胞(BMSCs)的增殖及其相关机制.方法 体外培养BMSCs,应用流式细胞术鉴定BMSCs,用脂多糖(LPS)刺激BMSCs,应用CCK-8法检测BMSCs的增殖能力;采用Western印迹检测大鼠BMSCs中TLR4蛋白表达;实验分为对照组、LPS组、TLR4阻断剂+LPS组、Wnt通路经典抑制剂(DKK-1) +LPS组.应用实时荧光定量PCR法检测BMSCs中β-catenin、c-myc及细胞周期蛋白(cyclin) D1 mRNA表达,应用Western 印迹检测蛋白表达.结果 与对照组相比,LPS组BMSCs的细胞增殖能力明显增强,TLR4呈高表达(均P＜0.01);LPS组β-catenin、c-myc和cyclinD1表达显著增加(P＜0.01).与LPS组相比,加入TLR4阻断剂后,β-catenin、c-myc和cyclinD1表达显著下调(P＜0.05,P＜0.01),加入DDK-1后,β-catenin、c-myc和cyclinD1表达显著降低(均P＜0.01).结论 TLR4促进BMSCs的增殖,其机制可能与调节Wnt/β-catenin信号通路有关.%Objective To investigate the effect of Wnt/beta-catenin signaling pathway activated by Toll like receptor (TLR) 4 on the proliferation of mesenchymal stem cells (MSCs) and the mechanisms.Methods MSCs were cultured in vitro.Flow cytometry was used to identify MSCs.The experiment was divided into control,LPS,LPS +TLR4 blockers,LPS+DKK-1 groups.Cell proliferation was evaluated by CCK-8 assay.The mRNA and protein expressions of β-catenin,c-myc,cyclinD1 were detected by RT-PCR and Western blot,respectively.Results Compared to that of control group,cell proliferation was significantly increased in LPS group (P＜0.01).LPS treatment increased the expressions of TLR4,β-catenin,c-myc and cyclinD1 in MSCs(P＜0.01).Compared to LPS group,TLR4 blockers and inhibitor DDK-1 decreased the expressions of β-catenin,c-myc and cyclinD1,respectively(P＜0.01).Conclusions TLR4 promotes the proliferation of MSCs,and its mechanism is probably related to the up-regulation of Wnt/beta-catenin signaling pathway.
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