白藜芦醇通过抑制β-catenin表达诱导并促进人肌腱干细胞的成骨分化

Resveratrol induce and promote the osteogenic differentiation of human tendon stem cells via inhibiting β-catenin expression

[目的]观察白藜芦醇对人肌腱干细胞成骨分化的作用及其细胞信号通路。[方法]低密度接种法和胶原酶消化法分离培养人肌腱干细胞,结晶紫染色观察分离出的细胞形成的单克隆,体外诱导检测分离细胞的多向分化能力;在/无成骨诱导因子下,不同浓度白藜芦醇刺激人肌腱干细胞荧光定量PCR检测骨相关基因(Run x2和OCN)表达,免疫印迹检测Run x2蛋白表达,茜素红S染色观察钙盐沉积,免疫印迹检测β-catenin和NK-KB表达以检测相关细胞信号通路。[结果]成功从人髌腱组织中分离培养出肌腱干细胞,体外可被诱导成骨细胞和脂肪细胞;在/无成骨诱导因子影响下,白藜芦醇均促进骨相关因子Run x2和其下游因子OCN表达,且随着白藜芦醇的浓度升高而上调;无成骨诱导因子影响下,Run x2蛋白表达随着白藜芦醇浓度升高而增多,而在成骨诱导因子存在下,其蛋白表达变化不明显。在/无成骨诱导因子影响下,茜素红S染色显示白藜芦醇促进人肌腱干细胞的钙盐沉积。随着白藜芦醇浓度的提高,β-catenin表达减少,而NF-KB表达无明显变化。[结论]白藜芦醇可以体外诱导并促进人肌腱干细胞的成骨分化,可能通过调节β-catenin信号通路来调控。

[Objective] To observe the effect of resveratrol on osteogenic differentiation of human tendon stem cells.[Methods] The tendon stem cells were isolated from human patellar tendon in very low seeded density after collagenase digest. The multi- differentiation potential of isolated tendon cells was measured by induced medium. Different concentrations of resveratrol was added in tendon stem cells with completed medium or osteogenic medium. q PCR was used to detect the run x2 and OCN gene level,Western blot was used to detect the run x2 / β- catenin / NF- KB / β- actin expression,alizarin red s staining was used to observe the mineral deposition. [Results] Human tendon stem cells were isolated from patellar tendon,isolated tendon cells could differentiate into osteocytes and adipocytes in vitro. The Run x2 and OCN mRNA level was upregulated by resveratrol in complete medium and osteogenic medium. Resveratrol promoted Run x2 expression in complete medium but not in osteogenic medium. Resveratrol induced mineral deposition in complete medium and promoted in osteogenic medium. The β- catenin expression was downregulated by resveratrol,while the NF- KB expression was unchanged. [Conclusion] Resveratrol inducing osteogenic differentiation in complete medium and promoting in osteogenic medium maybe via inhibiting β- catenin cell signal pathways.