Liu F, Malaval L, Gupta A K, Aubin J E
Medical Research Council Group in Periodontal Physiology, University of Toronto, Ontario, Canada.
Dev Biol. 1994 Nov;166(1):220-34. doi: 10.1006/dbio.1994.1309.
Messenger RNA expression analyzed by in situ hybridization and Northern analysis and protein expression analyzed biochemically or immunocytochemically have been used to study the developmental expression of various osteoblast (OB)-associated molecules. These approaches have shown that over a time course of OB differentiation in vivo and in vitro, the expression of macromolecules associated with OB cells changes. However, ambiguities in data from different approaches and in populations representative of cells at different developmental stages are extant. To begin to discriminate differentiation stages with more precision and to address intercellular heterogeneity, fetal rat calvaria cells were grown at low densities under conditions in which bone nodules form and mineralize and colonies were classified morphologically as fibroblastic or osteoblastic (early, intermediate, or mature). Whole discrete colonies and single cells from individual colonies were analyzed molecularly by a random amplification poly(A)-polymerase chain reaction (PCR) and for protein expression by immunocytochemistry; we analyzed the expression of known bone-related macromolecules (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin). Both PCR and immunocytochemistry revealed that different colony types were reproducibly distinguishable in their expression of either general (collagen type I) or bone-associated (alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin) macromolecules, such that fibroblastic colonies were distinguishable from osteoblastic colonies and the latter could be subdivided into less mature or more mature osteoblastic colonies. While some aspects of the temporal differentiation sequence defined earlier were confirmed, several additional features were evident from these single cell-single colony studies. First, different repertoires of OB-associated markers were expressed in different cells, suggesting variation in the switch-on of the OB differentiation program and heterogeneity in the OB phenotype. Second, among colonies classified as fibroblastic on the basis of morphology heterogeneity was also evident and there were some cells expressing features consistent with their being osteoprogenitor cells. Our data support the hypothesis that individual fibroblastic and osteoblastic cells are heterogeneous in expression of marker molecules. We also conclude that individual cells and colonies analyzed by poly(A)-PCR will be useful in lieu of mass populations to extend investigation of stages in the progression of OB differentiation.
通过原位杂交和Northern分析来分析信使核糖核酸的表达，以及通过生化或免疫细胞化学方法来分析蛋白质表达，已被用于研究各种成骨细胞（OB）相关分子的发育表达。这些方法表明，在体内和体外OB分化的时间过程中，与OB细胞相关的大分子表达会发生变化。然而，来自不同方法的数据以及代表不同发育阶段细胞群体的数据中存在模糊性。为了更精确地区分分化阶段并解决细胞间的异质性问题，将胎鼠颅骨细胞在低密度条件下培养，在这种条件下骨结节形成并矿化，并且将菌落根据形态学分类为成纤维细胞型或成骨细胞型（早期、中期或成熟）。通过随机扩增聚腺苷酸聚合酶链反应（PCR）对整个离散菌落和单个菌落中的单个细胞进行分子分析，并通过免疫细胞化学分析蛋白质表达；我们分析了已知的骨相关大分子（I型胶原蛋白、碱性磷酸酶、骨桥蛋白、骨唾液蛋白和骨钙素）的表达。PCR和免疫细胞化学均显示，不同菌落类型在一般（I型胶原蛋白）或骨相关（碱性磷酸酶、骨桥蛋白、骨唾液蛋白和骨钙素）大分子的表达上具有可重复性的差异，使得成纤维细胞菌落与成骨细胞菌落可区分开来，并且后者可细分为不太成熟或更成熟的成骨细胞菌落。虽然早期定义的时间分化序列的某些方面得到了证实，但这些单细胞-单菌落研究还发现了几个额外的特征。首先，不同的OB相关标志物在不同细胞中表达，这表明OB分化程序的开启存在差异，并且OB表型存在异质性。其次，在基于形态学分类为成纤维细胞型的菌落中也明显存在异质性，并且有一些细胞表达的特征与它们作为骨祖细胞一致。我们的数据支持这样的假设，即单个成纤维细胞和成骨细胞在标志物分子的表达上是异质的。我们还得出结论，通过聚腺苷酸PCR分析的单个细胞和菌落将有助于代替大量细胞群体，以扩展对OB分化进程中各阶段的研究。
