不同浓度淫羊藿苷对大鼠脂肪干细胞成骨分化相关因子的体外研究

Effect of icariin on osteogenic differentiation of rat adipose-derived stem cells in vitro

目的探究淫羊藿苷对大鼠脂肪干细胞成骨分化的影响。方法分离、培养SPF级SD雌性大鼠脂肪干细胞后,流式细胞术进行表面标志物检测,然后通过茜素红、油红O染色观察脂肪干细胞的成骨分化及成脂分化。将第3代脂肪干细胞分为空白组、白藜芦醇组(100μmol/L)、淫羊藿苷Ⅰ~Ⅳ组(1、10、50、100μmol/L),培养21 d后对脂肪干细胞LIM结构域蛋白Ajuba(Ajuba)、Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)进行荧光定量RT-PCR、Western-blotting检测。结果培养至第3代的脂肪干细胞表面标志物表现为CD90(95%)、CD34(9.26%)。成骨诱导21 d时茜素红染色可见橘红色钙化结节,成脂诱导21 d时油红O染色可见红色脂滴。白藜芦醇与100μmol/L淫羊藿苷均能促进脂肪干细胞Runx2、ALP基因及蛋白表达,且与白藜芦醇比较,同浓度的淫羊藿苷呈现出显著的促进效应(P<0.01,P<0.05)。白藜芦醇及各浓度淫羊藿苷均可抑制Ajuba基因及蛋白表达,且与100μmol/L白藜芦醇比较,50、100μmol/L淫羊藿苷抑制Ajuba基因表达差异无统计学意义,10、50、100μmol/L淫羊藿苷抑制Ajuba蛋白表达差异均无统计学意义。结论淫羊藿苷与白藜芦醇均具有促进大鼠脂肪干细胞成骨分化以及抑制其成脂分化的作用,二者抑制成脂分化的效应相当,而淫羊藿苷对成骨分化的促进作用优于同浓度的白藜芦醇。

Objective We aimed to investigate the effect of icariin on osteogenic differentiation of rat adipose stem cells.Methods Adipose stem cells from SPF female Sprague-Dawley rats were isolated and cultured.Surface markers were detected,and the osteogenic and adipogenic differentiation were determined by alizarin red and oil red O staining.Third-passage adipose stem cells were divided into the blank group,the resveratrol group(100μmol/L),and the icariinⅠtoⅣgroups(1,10,50,and 100μmol/L).After 21 days of culture,the mRNA and protein expression levels of the LIM domain protein Ajuba(Ajuba),Runt-related transcription factor 2(Runx2),and alkaline phosphatase(ALP)were determined by qRT-PCR and Western blotting analysis,respectively.Results Of the cultured third-passage adipose stem cells,95%were positive for the surface marker CD90 and 9.26%were positive for CD34.Orange calcified nodules could be observed after alizarin red staining after 21 days of osteogenic induction,and red lipid droplets could be observed after oil red O staining after 21 days of adipogenic induction.Both resveratrol and 100μmol/L icariin could promote the mRNA and protein expressions of Runx2 and ALP in adipose stem cells,and compared with resveratrol,the same concentration of icariin showed a significant promoting effect(P<0.01).Both resveratrol and icariin could inhibit the mRNA and protein expression of Ajuba.There was no significant difference in Ajuba mRNA and protein expression in the 50μmol/L and 100μmol/L icariin groups compared with the 100μmol/L resveratrol group,and there was no significant difference in Ajuba protein expression in the 10,50,and 100μmol/L icariin groups.Conclusion Icariin and resveratrol can promote osteogenic differentiation and inhibit adipogenic differentiation of rat adipose stem cells.They had the same inhibitory effect on adipogenic differentiation,while icariin had a stronger promoting effect on osteogenic differentiation than resveratrol.