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目的探讨白藜芦醇(resveratrol,RES)对软骨前体细胞ATDC5肥大化的影响及其可能机制。方法通过CCK-8法研究不同浓度RES对ATDC5细胞活性的影响。建立软骨肥大化诱导体系,根据活性实验的安全浓度,设立对照组(0μmol/L)和RES组(1μmol/L)。qRT-PCR和Western blot法确定肥大化相关因子RUNX2、MMP13和COLX表达变化;甲苯胺蓝和免疫组化分析细胞外基质粘多糖及COLX、RUNX2表达;利用qRT-PCR检测软骨细胞肥大化经典通路WNT、IHH和FGF中相关因子的表达变化。结果 RES安全使用浓度为1μmol/L;与对照组相比,在肥大化诱导14 d后,RES组ATDC5细胞肥大化水平明显提升;RES组肥大化相关基因表达量在3、7、14 d时明显增加;WNT和IHH途径中促肥大化因子β-catenin和GLI2的表达水平在RES组较对照组升高,而GLI3表达水平下降。FGF途径中PI3K的表达水平较对照组没有变化。结论 RES通过激活β-catenin及GLI2,抑制GLI3,促进RUNX2表达,实现促进ATDC5肥大化的目的。
Objective To investigate the effect of resveratrol (RES) on the hypertrophy of cartilage progenitor cells (ATDC5) and its possible mechanism. Methods The effects of different concentrations of RES on the activity of ATDC5 cells were studied by CCK-8. The inducing system of cartilage hypertrophy was established. According to the safe concentration of active test, the control group (0μmol / L) and the RES group (1μmol / L) were established. qRT-PCR and Western blot were used to determine the expressions of RUNX2, MMP13 and COLX, respectively. Toluidine blue and immunohistochemistry were used to analyze the expression of extracellular matrix mucopolysaccharide and COLX, RUNX2. qRT-PCR was used to detect the chondrocyte hypertrophy pathway WNT, IHH and FGF expression changes in relevant factors. Results The safe use concentration of RES was 1 μmol / L. Compared with the control group, the hypertrophy of ATDC5 cells in RES group was significantly increased after 14 days of inducement; And the expression of pro-hypertrophic factors β-catenin and GLI2 in WNT and IHH pathway were higher in RES group than in control group, while GLI3 expression level decreased. The level of PI3K in FGF pathway was not changed compared with control group. Conclusion RES can inhibit GLI3 and promote RUNX2 expression by activating β-catenin and GLI2, and achieve the goal of promoting the hypertrophy of ATDC5.