老鹳草素通过Wnt/β-catenin信号通路影响小鼠骨髓基质干细胞的增殖和成骨分化

Effect of Geraniin Mediated Wnt/β-catenin Signal Pathway on Proliferation and Osteogenic Differentiation of Mice Bone Marrow Mesenchymal Stem Cells

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。

Abstract：Objective：To investigate the role of Wnt/β -eatenin signaling pathway playing in the induction effect of ge- raniin on proliferation and differentiation of murine bone marrow mesenchymal stem cells（ BMSCs）into osteoblasts. Methods ：BMSCs were isolated and cultured from mice long bones. Six groups were designed, control group （DMEM/F12 com- plete medium）, geraniin low - dose group （ 10^-9 mol·L^-1 geraniin ＋ DMEM/F12 complete medium）, geraniin middle - dose group（ 10^-8 mol·L^-1 geraniin ＋ DMEM/F12 complete medium） ,geraniin high- dose group（ 10^-7mol·L^-1 geraniin ＋ DMEM/F12 complete medium） ,geraniin high - dose ＋ DKK1 （ Wnt/β - catenin signaling pathway specific inhibi- tor） group and DKK1 group. After 7 days of osteogenic induction,CCK -8 was used to detect the proliferation of BMSCs. The activity of alkaline phosphatase（ALP） and osteocalcin（ OCN ） content were assayed. After 48 h of osteogenic induc- tion, Real time -PCR and Western- blot were used to detect the expression of the critical signaling molecules in Wnt/β - catenin signaling pathway （ Wnt3 a, β - catenin, GSK - 3 β, Axin2 and Runx2）. Results ： After 7 days of osteogenic induc- tion,geraniin treatment dose - dependently promoted the proliferation of BMSCs compared with control group （all P 〈 0. 05）. Meanwhile, ALP activity and OCN content were significantly increased compared with those of the control group （ βall P 〈 0.05 ）. After 48 h of osteogenic induction, geraniin treatment dose - dependently decreased the expression of GSK- β and promoted the expression of Wnt3a, β-catenin,Axin2 and Runx2 in bone marrow mesenchymal stem ceils under osteogenic induction（ all P 〈 0. 05）. In experimental group 5 （ with DKK1 ）, cell viability, ALP activity, OCN content and expression levels of Wnt3a, β -catenin, Axin2 and Runx2 decreased significantly compared with those of experiment group 4 while the expression level of GSK - 3β increased significantly compared with that of experiment group 4 （ all P 〈0. 05）. Conclusion： Geraniin promotes the proliferation and osteogenic differentiation of BMSCs, in which Wnt/β - catenin signaling pathway might be involved.