miR-34b对骨髓间充质干细胞成骨分化的影响及相关机制

Effect of miRNA-34b on osteogenic differentiation of h BMSCs and its protential mechanism

目的:探讨mi RNA-34b在骨髓间充质干细胞成骨分化过程中的表达及其可能的作用靶点和作用机制。方法:采用密度梯度离心和全骨髓贴壁相结合的方法分离培养人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,h BMSCs),并在体外诱导成骨分化。采用实时荧光定量PCR技术,检测h BMSCs成骨分化过程中的mi R-34b的表达水平;然后过表达mi R-34b,进一步观察其对h BMSCs成骨分化的影响。同时检测过表达mi R-34b对成骨分化的关键信号通路之一Notch信号通路的活性影响,初步探讨其可能涉及的作用机制。结果:成功分离出h BMSCs,并构建了h BMSCs体外诱导成骨分化模型;且随着成骨诱导培养时间的延长,mi RNA-34b表达水平逐渐降低。ALP活性检测、茜素红染色检测结果显示过表达mi RNA-34b后,ALP活性显著降低,且茜素红染色的钙盐结节明显减少;同时Western blot实验结果显示过表达mi RNA-34b后,成骨特异性标记分子Runx2的蛋白表达水平显著下降(P<0.05)。此外,过表达mi RNA-34b后,Notch信号通路的活性显著降低。结论:mi RNA-34b能够负向调控人骨髓间充质干细胞成骨分化;其作用机制可能与抑制Notch信号通路的活性有关,提示mi RNA-34b可以作为诊断和靶向治疗慢性炎症性骨疾病的潜在作用靶点。

Objective： To investigate the expression of miRNA-34b and its effect and mechanism on osteogenic differentiation of human bone marrow mesenchymal stem cells （hBMSCs）.Methods： hBMSCs were isolated and cultured by the improved density gradient centrifugation combined with whole bone marrow adherent method, and cells were induced into osteoblasts by cultivation in osteogenic differentiation medium. The expression of miRNA-34b was detected by qRT-PCR and Western blot. hTen miRNA-34b was overexpressed by transfection with miRNA-34b mimics and detected its influence on osteogenic differentiation of hBMSCs; meanwhile Western blot was used to analysis the activity of Notch pathway, in order to investigating its potential mechanism. Results：hBMSCs were successfully isolated and cultured in vitro and could be stably induced into osteoblasts when subjected to osteogenic differentiation medium. miR-34b expression decreased during the osteogenic differentiation of hBMSCs. Overexpression of miR-34b could suppress the ALP activity, mineral nodule formation and down regulate the osteogenic differentiation marker Runx2 （P＜0.05）. Furthermore, overexpression of miR-34b could suppress Notch pathway.Conclusion： miR-34b down regulates the osteogenic differentiation of hBMSCs and it is probably connected to the suppression of Notch pathway, which means that miR-34b could be a potential target for diagnosis and treatment of chronic inlfammatory bone diseases.