颅骨锁骨发育不全患者骨髓基质细胞的增殖、成骨和衰老

›› 2015, Vol. 6 ›› Issue (2): 66-70.

颅骨锁骨发育不全患者骨髓基质细胞的增殖、成骨和衰老

张娟¹,戈杰¹,李光南¹,周培培¹,江宏兵²

1. 江苏省南京医科大学附属口腔医院口腔颌面外科
2. 南京医科大学口腔医学院

收稿日期:2015-03-23 修回日期:2015-06-05 出版日期:2015-06-25 发布日期:2015-07-13
通讯作者: 江宏兵 E-mail:jhbcd@sina.com
基金资助:
国家自然科学基金;江苏高校优势学科建设工程资助项目

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

1. Department of Oral and Maxillofacial Surgery, School of Stomatology, Nanjing Medical University
2.

Received:2015-03-23 Revised:2015-06-05 Online:2015-06-25 Published:2015-07-13

摘要/Abstract

摘要： 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 factor 2，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。

Abstract: Objective：To study the in vitro biologic characteristics of bone marrow stromal cells with cleidocranial dysplasia (BMSCs-CCD), including osteogenesis, proliferation ability, stemness and senescence. Methods：MTT and cell cycle detection for proliferation 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 experiment 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 senescenced 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.

张娟戈杰李光南周培培江宏兵. 颅骨锁骨发育不全患者骨髓基质细胞的增殖、成骨和衰老[J]. , 2015, 6(2): 66-70.

参考文献

[1]Mundlos S, Otto F, Mundlos C, et al.Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia[J].Cell,1997,89(5):773-779.
[2]Baumert U, Golan I, Redlich M, et al.Cleidocranial dysplasia: molecular genetic analysis and phenotypic-based description of a Middle European patient group[J].Am J Med Genet A,2005,139A(2):78-85.
[3]Morrison NA, Stephens AA, Osato M, et al.Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation[J].PLoS One,2012,7(8):e42617-.
[4]Zanatta M, Valenti MT, Donatelli L, et al.Runx-2 gene expression is associated with age-related changes of bone mineral density in the healthy young-adult population[J].J Bone Miner Metab,2012,30(6):706-714.
[5]Saeed H, Iqtedar M.Bone Marrow Stromal Cell (BMSC) and skeletal aging: role of telomerase enzyme[J].Pak J Pharm Sci,2014,27(2):321-333.
[6]Dalle Carbonare L, Innamorati G, Valenti MT.Transcription factor Runx2 and its application to bone t3 engineering[J].Stem Cell Rev,2012,8(3):891-897.
[7]Ding B, Li C, Xuan K, et al.The effect of the cleidocranial dysplasia-related novel 1116_1119insC mutation in the RUNX2 gene on the biological function of mesenchymal cells[J].Eur J Med Genet,2013,56(4):180-187.
[8]戈杰，张娟，郭松松, 等.颅骨锁骨发育不全患者牙囊细胞的体外生物学特征[J].口腔生物医学,2014,5(4):169-173.
[9]Pantovic A1, Krstic A, Janjetovic K, et al.Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells[J].Bone,2013, 52(1):524-531.
[10]Chen H, Ghori-Javed FY, Rashid H, et al.Runx2 regulates endochondral ossification through control of chondrocyte proliferation and differentiation[J].J Bone Miner Res,2014,29(12):2653-2665.
[11]Tsuji K, Komori T, Noda M.Aged mice require full transcription factor, Runx2/Cbfa1, gene dosage for cancellous bone regeneration after bone marrow ablation[J].J Bone Miner Res,2004,19(9):1481-1489.
[12]Vaughan T, Pasco JA, Kotowicz MA, et al.Alleles of RUNX2/CBFA1 gene are associated with differences in bone mineral density and risk of fracture[J].J Bone Miner Res,2002,17(8):1527-1534.
[13]Dong W, Zhang P, Fu Y, et al.Roles of SATB2 in site-specific stemness, autophagy and senescence of bone marrow mesenchymal stem cells[J].J Cell Physiol,2015,230(3):680-690.
[14]Abdallah BM, Jafari A, Zaher W, et al.Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation[J].Bone,2015,70:28-36.