摘要
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.
出处
《中华中医药学刊》
CAS
北大核心
2017年第1期215-218,共4页
Chinese Archives of Traditional Chinese Medicine
基金
河北省中医药管理局科研计划项目(2015137)