骨碎补总黄酮对iPS⁃MSCs成骨分化的影响

Effects of drynaria flavonoids on the osteogenic differentiation of iPS-MSCs

目的研究骨碎补总黄酮对诱导多能干细胞来源的间充质干细胞(iPS-MSCs)成骨分化的影响。方法将骨碎补总黄酮稀释为15.63、31.25、62.5、125、250μg/mL的6个浓度,加入细胞培养基中,其中不含骨碎补总黄酮的培养基作为对照组,将iPS-MSCs分别接种于不同浓度的培养基中。采用CCK8法检测各组iPS-MSCs增殖情况,ALP活性检测及茜素红染色法检测各组成骨分化情况,RT-PCR检测各组OCN、CollagenⅠmRNA表达情况。结果CCK8法检测显示125、250μg/mL浓度的骨碎补总黄酮抑制iPS-MSCs生长,因此选用62.5μg/mL浓度以下研究iPS-MSCs的分化情况。在成骨诱导培养基培养后,3个处理组ALP活性均高于空白对照组,并伴有茜素红观察钙化结节相应增多,差异均具有统计学意义(P<0.01)。除此之外,RT-PCR结果显示3个浓度组的OCN、CollagenⅠmRNA表达量均高于空白对照组,差异具有统计学意义(P<0.01)。结论骨碎补总黄酮能够促进iPS-MSCs成骨分化,其作用机制可能与促成骨关键因子OCN和collagenⅠ的表达有关。

Objective To study the effect of total flavonoids of rhizoma drynariae on osteogenic differentiation of mesenchymal stem cells(MSCs)derived from induced pluripotent stem cells(iPSCs).Methods The total flavonoids of rhizoma drynariae were diluted to concentrations of 15.63,31.25,62.5,125,and 250μg/mL,respectively,and added to the cell culture medium.The medium did not contain total flavonoids was as the control group.iPS-MSCs were inoculated into different concentrations of culture medium.CCK8 method was used to detect the proliferation of iPS-MSCs in each group.ALP activity detection and Alizarin red staining method were used to detect the bone differentiation of each components.RT-PCR was used to detect the mRNA expressions of OCN and collagen I in each group.Results CCK8 result showed that the total flavonoids of rhizoma drynariae at the concentration of 125μg/mL and 250μg/mL inhibited the growth of iPS-MSCs.Therefore,the concentration of 62.5μg/mL or less was selected to study the differentiation of iPS-MSCs.After cultured in osteogenic induction medium,the activity of ALP in the three treatment groups was higher than that in the 0μg/mL blank control group,the calcified nodules were increased with Alizarin red staining,and the difference was statistically significant(P<0.01).In addition,RT-PCR result showed that the mRNA expressions of OCN and collagen I in the three concentration groups were significantly higher than those in the control group.Conclusion Total flavonoids of rhizoma drynariae promotes the osteogenic differentiation of iPS-MSCs.Its mechanism may be related to the increased expressions of OCN and collagen I,the key factors for osteogenesis.