颅骨锁骨发育不全患者骨髓基质细胞的增殖、成骨和衰老

Study of proliferation,osteogenesis and senescence of bone marrow stromal cells from a cleidocranial dysplasia patient

目的:骨髓基质细胞(bone marrow stromal cells,BMSCs)在调节颅骨锁骨发育不全(cleidocranial dysplasia,CCD)患者骨结构中发挥关键作用,本研究通过与正常BMSCs比较分析,探讨CCD患者BMSCs的体外增殖、成骨分化、干性及衰老特征。方法:分离培养CCD患者及正常同龄人BMSCs;甲基噻唑基四唑(MTT)法及流式细胞周期分析其增殖能力;成骨诱导后采用Western blot及茜素红染色分析其成骨能力;检测多潜能转录因子及克隆形成,分析其干性能力;检测衰老调控关键基因p16、p21表达及通过β-gal衰老染色分析其衰老特征。结果:与正常BMSCs相比,BMSCs-CCD增殖活性低,且细胞周期中处于S期、G2的细胞比例较低;两组细胞成骨诱导1、3、7 d后,BMSCs-CCD组中Runt相关转录因子2(Runt-related transcription factor2,Runx2)、成骨细胞特异性转录因子Osterix、骨桥蛋白(Osteopontin,Opn)表达水平较正常组低;成骨诱导14 d后,BMSCs-CCD组形成的钙化结节较正常组少;BMSCs-CCD组中多潜能转录因子Oct4、Nanog、Sox2表达及克隆形成率均较正常组低;相反,BMSCs-CCD组中p16、p21等衰老标记分子表达及衰老细胞阳性染色比例均较正常组高。结论:同正常BMSCs比较,CCD患者BMSCs的增殖能力、成骨能力、干性强度均较差,且更易衰老。这些特征可能是CCD患者易发骨质疏松及骨折的生物学机制之一。

Objective：To study the in vitro biologic characteristics of bone marrow stromal cells with cleidocranial dysplasia （BM-SCs-CCD）,including osteogenesis,proliferation ability,stemness and senescence.Methods：MTT and cell cycle detection for prolifera-tion ability,Western blot and alizarin red staining for osteogenesis ability,Western blot and clony-formation for stemness ability,Western blot and senescence staining for senescence characteristics.Results：The optical density value was lower in BMSCs-CCD in MTT experi-ment and lower BMSCs-CCD were under S or G2 stage while more under G1 stage in cell cycle detection.BMSCs-CCD expressed lower levels of Runx2,Opn,Osterix and weaker alizarin red staining.Moreover,BMSCs-CCD also exhibited weaker stemness associate proteins （Oct4,Nanog,and Sox2 ）and clony-formation rates.However,stronger senescence associated proteins （p16,p21 ）and more senes-cenced cells were found in BMSCs-CCD.Conclusions：BMSCs-CCD exhibited weaker proliferation ability,osteogenesis and stemness while stronger senescence ability as compared to BMSCs.These results are helpful for us to understand pathological mechanism of CCD bone disease.