目的：探讨组蛋白去甲基化酶JMJD3对骨髓基质干细胞（bone marrow stromal cells，BMSCs）体外多潜能性及成骨分化的生物学调控作用。方法：用离心和贴壁培养相结合方法分离培养大鼠四肢骨BMSCs，分别用含JMJD3-shRNA的绿色荧光蛋白（GFP）慢病毒干扰载体（实验组）及含无关序列的GFP慢病毒载体（对照组）转染BMSCs，Western blot方法检测两组BMSCs中组蛋白H3第27位赖氨酸上三甲基化（H3K27me3）及干细胞多潜能转录因子Oct4、Nanog、Sox2的表达，并比较两组细胞的克隆形成率；实时定量RT-PCR、Western blot、茜素红染色检测两组细胞的体外成骨分化能力。结果：与对照组相比，实验组BMSCs的H3K27me3表达水平升高，具有更强的克隆形成能力，且表达更强的Oct4、Nanog、Sox2等多潜能因子；体外成骨诱导7 d后，实验组BMSCs中RUNX2等成骨分化基因表达下降，诱导14 d后，钙结节形成较对照组少。结论：抑制JMJD3表达可增强BMSCs的干性特征，抑制其成骨分化。%Objective:To investigate the role of the histone demethylases JMJD 3 in regulating the pluripotency and the osteogenic differentiation of the bone marrow stromal cells (BMSCs)in vitro.Methods:Gradient centrifugation and adherent culture were used to isolate the BMSCs from rat tibia, and the lenti-virus of the JMJD3-shRNA and GFP (as the control) were constructed.The expression of H3K27me3 and the pluripotent transcription factors ( Oct4/Nanog/Sox2) were detected by Western blot .The colony-forming effi-ciency was compared between JMJD 3-shRNA group and GFP group .The different osteogenic ability of the JMJD 3-shRNA group and the control group were analyzed by real-time RT-PCR, Western blot and Alizarin red staining after osteogenic induction .Results:Com-pared with the control, the expression of H3K27me3 was higher in JMJD3-shRNA group.The JMJD3 shRNA group possessed stronger colony-forming ability, expressed higher stemness markers (Oct4/Nanog/Sox2), compared with the control group .After 7-day osteo-genic induction in vitro , the BMSCs′osteogenic ability declined in JMJD 3-shRNA group .After 14-day osteogenic induction , the calci-um nodules of JMJD3-shRNA group were less than that of the control group .Conclusions:Inhibition of JMJD3 can enhance BMSCs′stemness, and suppress their osteogenic differentiation .
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