论文部分内容阅读
目的:观察Wnt/β-catenin信号通路在老鹳草素诱导小鼠骨髓基质干细胞(BMSCs)增殖和成骨分化中的作用机制。方法:分离、培养小鼠BMSCs,分为对照组(DMEM/F12完全培养基)、老鹳草素低剂量组(10-9mol·L-1老鹳草素+DMEM/F12完全培养基)、老鹳草素中剂量组(10-8mol·L-1老鹳草素+DMEM/F12完全培养基)、老鹳草素高剂量组(10-7mol·L-1老鹳草素+DMEM/F12完全培养基)、老鹳草素高剂量+DKK1(Wnt/β-catenin信号通路特异性阻断剂)组和DKK1组。成骨诱导第7天时,采用CCK-8法检测细胞增殖,碱性磷酸酶(ALP)、骨钙素(OCN)试剂盒测定ALP活性及OCN含量。成骨诱导48 h时,采用Real time-PCR和Western-blot检测各组Wnt/β-catenin信号通路的关键信号分子Wnt3a、β-catenin、GSK-3β、Axin2、Runx2 mRNA和蛋白表达。结果:成骨诱导第7天时,老鹳草素各剂量组明显促进BMSCs的增殖,呈剂量依赖性(均P<0.05);同时ALP活性及OCN含量较对照组显著升高,呈剂量依赖性(均P<0.05)。成骨诱导48 h时,老鹳草素各剂量组Wnt3a、β-catenin、Axin2、Runx2 mRNA和蛋白表达较对照组显著上调,GSK-3βmRNA和蛋白表达较对照组显著下调,呈剂量依赖性(均P<0.05)。加入DKK1后,细胞增殖受到显著抑制,ALP活性及OCN含量显著降低(均P<0.05);同时Wnt3a、β-catenin、Axin2、Runx2 mRNA和蛋白表达显著下调,GSK-3βmRNA和蛋白表达显著上调(均P<0.05)。结论:老鹳草素能够明显促进BMSCs的增殖及向成骨方向分化,其机制与Wnt/β-catenin信号通路的激活有关。
AIM: To investigate the mechanism of Wnt / β-catenin signaling pathway in the proliferation and osteogenic differentiation of mouse bone marrow stromal stem cells (BMSCs) induced by Geranium. Methods: BMSCs were isolated and cultured and divided into control group (DMEM / F12 complete medium), geraniol low dose group (10-9mol·L-1 safranin + DMEM / F12 complete medium) Geranium Suginotonic medium dose group (10-8mol·L-1 geranium + DMEM / F12 complete medium), high-dose geraniol group (10-7mol·L-1 geraniol + DMEM / F12 complete medium), high dose of geraniol + DKK1 (Wnt / β-catenin signaling pathway-specific blocker) group and DKK1 group. On the 7th day after osteogenic induction, the cell proliferation, alkaline phosphatase (ALP) and osteocalcin (OCN) were detected by CCK-8 assay for ALP activity and OCN content. At 48 h after osteogenic induction, the mRNA and protein expressions of Wnt3a, β-catenin, GSK-3β, Axin2 and Runx2 in Wnt / β-catenin signaling pathways were detected by Real time-PCR and Western-blot. RESULTS: On the 7th day after osteogenic induction, each dose of geraniol significantly promoted the proliferation of BMSCs in a dose-dependent manner (all P <0.05). At the same time, ALP activity and OCN content were significantly increased compared with the control group (All P <0.05). At 48 h after osteogenic induction, the mRNA and protein expressions of Wnt3a, β-catenin, Axin2 and Runx2 in each dose of geranyltin were significantly up-regulated compared with the control group, and the expressions of GSK-3β mRNA and protein were significantly decreased compared with the control group All P <0.05). After addition of DKK1, cell proliferation was significantly inhibited, ALP activity and OCN content were significantly decreased (all P <0.05); At the same time, the mRNA and protein expressions of Wnt3a, β-catenin, Axin2 and Runx2 were significantly downregulated and GSK-3βmRNA and protein expressions were significantly increased All P <0.05). Conclusion: Geranium could significantly promote the proliferation and osteogenic differentiation of BMSCs, and its mechanism is related to the activation of Wnt / β-catenin signaling pathway.