Frank Oliver, Heim Manuel, Jakob Marcel, Barbero Andrea, Schäfer Dirk, Bendik Igor, Dick Walter, Heberer Michael, Martin Ivan
Department of Surgery, Research Division, University of Basel, Switzerland.
J Cell Biochem. 2002;85(4):737-46. doi: 10.1002/jcb.10174.
We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.
我们开发并使用实时逆转录聚合酶链反应（RT-PCR）分析方法，来研究人骨髓基质细胞（BMSC）中典型成骨细胞相关基因的表达是如何受到以下因素调控的：（i）在诱导成骨分化的培养基中的培养时间；（ii）先前在增强细胞成骨能力的培养基中的扩增。来自六名健康供体的BMSC在不含（CTR）或含有成纤维细胞生长因子-2和地塞米松（FGF/Dex；已知这些因子可增强BMSC的成骨能力）的培养基中扩增，然后用抗坏血酸、β-甘油磷酸酯和地塞米松（这些因子通常用于诱导BMSC成骨分化）进一步培养长达20天。尽管不同个体之间基因表达水平存在很大差异，但我们确定了以下具有统计学意义的模式。在成骨培养基（OM）中，骨形态发生蛋白-2（BMP-2）、骨唾液蛋白-II（BSP）、骨桥蛋白（OP）以及程度较低的cbfa-1的mRNA水平随培养时间增加，在CTR和FGF/Dex扩增的BMSC中均如此，这与碱性磷酸酶、I型胶原、骨钙素和骨连接蛋白的水平不同。在OM中培养20天后，FGF/Dex扩增的BMSC中BMP-2、BSP和OP的表达高于CTR扩增的BMSC（mRNA水平分别高9.5倍、14.9倍和5.8倍），这与所有其他研究基因不同。对单克隆来源的BMSC菌株的分析进一步表明，在OM中培养20天后，只有一部分FGF/Dex扩增的克隆表达的BMP-2、BSP和OP的mRNA水平高于CTR扩增的克隆。总之，我们提供的证据表明，使用实时RT-PCR定量的BMP-2、BSP和OP的mRNA水平可作为监测体外BMSC成骨分化程度的标志物；使用这些标志物，我们进一步证明，FGF/Dex扩增后只有少数BMSC亚群表现出增强的成骨分化。
