富血小板纤维蛋白通过Wnt/β-catenin信号通路促进BMSCs成骨分化的研究

摘要

Objective: To investigate the effect of platelet-rich fibrin (PRF) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) via activating canonical Wnt/β-catenin signaling pathway. Methods: Rabbit BMSCs were isolated and identified. Blood samples were collected to prepare for PRF. Then PRF and BMSCs were co-culture with a transwell system. The experiments were divided into four groups:group A (BMSCs);group B (BMSCs/PRF);group C ( DKK1/BMSCs/PRF); group D (Wnt3a/BMSCs/PRF). After osteogenic differentiation for 3 weeks, the number of calcium nodule was identified by Alizarin red staining, and the ALP activity was detected by ALP kits. The expression of osteogenic and adipogenic markers (Runx2、OCN、PPARγ2 and LPL) were detected by qRT-PCR. Moreover, the expression of Wnt genes (CyclinD1 and β-catenin) were also examined. Results: Cells would be identified and confirmed their identity as BMSCs. Alizarin red staining showed that the number of calcium nodules in group B and group D were significantly more than in group A and C, and no significant difference between group B and group D were observed. The ALP activity in group B and group D were also significantly more than group A and group C. qRT-PCR results showed that the expression of osteogenic markers (Runx2、OCN) in group B and group D were significantly increased than group A and group C, but the expression of adipogenic markers (PPARγ2 and LPL) showed the opposite results. Furthermore, the expression of CyclinD1 and β-catenin in group B and group D were also significantly enhanced than group A and group C. Conclusion: PRF promotes osteogenic differentiation but inhibit adipogenic differentiation of BMSCs through activating canonical Wnt/β-catenin signaling pathway.%目的:研究富血小板纤维蛋白(PRF)通过Wnt/β-catenin信号通路对骨髓基质干细胞(BMSCs)分化的影响.方法:分离、培养原代兔BMSCs,取第三代细胞进行干细胞鉴定,收集同一只兔耳缘静脉血离心制备PRF,将PRF与BMSCs置于Transwell小室中共培养并进行成骨分化.实验分组:A组为单纯BMSCs对照组;B组为PRF与BMSCs共培养组;C组为加入DKK1的PRF与BMSCs共培养组;D组为加入Wnt3a的PRF与BMSCs共培养组.茜素红染色观察各组钙结节形成情况,碱性磷酸酶(ALP)试剂盒检测各组ALP活性,定量聚合酶链反应(qRT-PCR)检测成骨成脂相关基因Runx2、OCN、PPARγ2及LPL的mRNA表达,同时检测Wnt/β-catenin信号通路关键因子CyclinD1及β-catenin的mRNA表达.结果:流式细术检测结果显示,原代培养的BMSCs符合间充质干细胞鉴定标准.茜素红染色结果显示,B组和D组的钙结节数量明显多于A组和C组,A组和C组之间无明显差异.碱性磷酸酶(ALP)活性检测结果显示,B组与D组的ALP活性较A组和C组明显增加(P<0.05),且两组之间差异无明显统计学意义.qRT-PCR结果显示B组与D组中的成骨分化标志基因Runx2、OCN的mRNA表达,较A组和C组显著增加(P<0.05),而成脂分化标志基因PPARγ2、LPL的mRNA表达显著降低(P<0.05).此外,B组和D组中Wnt/β-catenin信号通路关键因子CyclinD1及β-catenin的mRNA表达较A组和C组显著增加(P<0.05).结论:PRF通过激活Wnt/β-catenin信号通路促进BMSCs成骨分化而抑制其成脂分化.