炎性环境中小白菊内酯对肌腱干细胞成骨分化的影响

Effect of parthenolide on osteogenic differentiation of tendon stem cells in inflammatory environment

[目的]观察炎性环境下小白菊内酯(PAR)对肌腱干细胞成骨分化的作用,探索其对骨肌腱接点损伤修复的作用及其信号通路。[方法]利用胶原酶法结合低密度接种法分离肌腱干细胞,体外验证分离细胞的成骨、成脂分化能力;TNF-α构建的炎性环境下,设置不同浓度组小白菊内酯(PAR)刺激人肌腱干细胞,用qRT-PCR检测炎症相关基因以及肌腱、成骨相关基因的表达,及Western Blot检测Run x2、β-catenin蛋白的表达以检测相关细胞信号通路。[结果]成功从人肌腱组织分离培养出干细胞。qRT-PCR检测显示,100 ng/ml TNF-α可引发炎症反应,PAR能抑制炎性相关基因COX-2、HIF-2α的表达,促进了成骨相关基因Run x2、OPN的表达,与TNF-α组对比差异有统计学意义(P<0.05);而肌腱相关基因Col I、Scleraxis的表达较复杂,与TNF-α组相比,下调了Col 1α水平,却上调Scleraxis水平。Western Blot检测结果显示,与TNF-α组对比,PAR显著增加蛋白Run x2、β-catenin的表达(P<0.05)。肌腱干细胞在体外可被诱导向成骨细胞和脂肪细胞分化。镜下观察茜素红S染色,PAR组均有红色钙盐沉积,相比之下对照组及TNF-α组基本无或出现少量钙盐沉积。[结论]炎性环境下小白菊内酯能控制TNF-α引起的炎性反应并可能通过β-catenin信号通路促进肌腱干细胞的成骨分化。

[Objective]To observed the effect of parthenolide(PAR)on osteogenic differentiation of tendon stem cells under the inflammatory environment created by TNF-α,and explore the signal pathway for repairing tendon-bone insertion injury.[Methods]Tendon stem cells were isolated by collagenase and seeded in a very low cell density.Capacity of multi-differentiation of the cells was verified by osteogenic and adipogenic induction in vitro.PAR in different concentration were added into the cell cultures to suppress the effect of TNF-α.qPCR and western blot were used to detect the expression of osteogenic genes and proteins.Furthermore,β-catenin cell pathway was also detected by western blot.Mineral deposits of tendon stem cells were stained by Alizarin Red S in order to observe the influence of PAR under inflammatory environment.[Results]The tendon stem cells were successfully isolated from human tendon tissue.As results of qPCR assay,the PAR significantly inhibited the expression of inflammation related genes COX-2 and HIF-2 e,while significantly enhanced expression of osteogenesis related genes Run x2 and OPN compared with the TNF-αgroup(P<0.05),however,the PAR had complex effects on the expression of tendon related gene Col I and Scleraxis,which upgraded expression of Col 1,whereas downgraded expression of Scleraxis compared with the TNF-αgroup.With respect of Western Blot assay,the PAR significantly increased the expression of RunX2 andβ-catenin in comparison with TNF-αgroup(P<0.05).The tendon stem cells were remarkably induced into osteocytes and adipocytes in vitro.Mineral deposits detected by Alizarin Red S staining were much more in groups with PAR,whereas no red mineral deposits were found in control or TNF-αgroup.[Conclusions]Under inflammatory environment created by TNF-α,parthenolide do inhibits the highly expressed inflammatory response and promoted osteogenic differentiation of tendon stem cells through theβ-catenin signaling pathway.