SIMULTANEOUS DETECTION OF MULTIPLE BONE-RELATED MESSENGER-RNAS AND PROTEIN EXPRESSION DURING OSTEOBLAST DIFFERENTIATION - POLYMERASE CHAIN-REACTION AND IMMUNOCYTOCHEMICAL STUDIES AT THE SINGLE-CELL LEVEL
SIMULTANEOUS DETECTION OF MULTIPLE BONE-RELATED MESSENGER-RNAS AND PROTEIN EXPRESSION DURING OSTEOBLAST DIFFERENTIATION - POLYMERASE CHAIN-REACTION AND IMMUNOCYTOCHEMICAL STUDIES AT THE SINGLE-CELL LEVEL
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DOI：
10.1006/dbio.1994.1309
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发表时间：
1994-11-01
影响因子：
2.7
通讯作者：
AUBIN, JE
中科院分区：
文献类型：
--
作者：
LIU, F;MALAVAL, L;AUBIN, JE
通过原位杂交和北方分析分析的信使RNA表达和生物化学或免疫细胞化学分析的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。这些方法已经表明，随着体内和体外OB分化的时间过程，与OB细胞相关的大分子的表达发生变化。然而，从不同的方法和在不同的发展阶段的细胞代表的群体中的数据的模糊性是现存的。为了开始更精确地区分分化阶段并解决细胞间异质性，在骨结节形成和矿化的条件下以低密度培养胎鼠颅骨细胞，并将集落在形态学上分类为成纤维细胞或成骨细胞（早期、中期或成熟）。通过随机扩增聚（A）-聚合酶链反应（PCR）和免疫细胞化学对整个离散集落和单个集落的单细胞进行分子分析;我们分析了已知骨相关大分子（I型胶原、碱性磷酸酶、骨桥蛋白、骨唾液蛋白和骨钙素）的表达。PCR和免疫细胞化学显示，不同的集落类型是可重复的区别，在其表达的一般（胶原I型）或骨相关的（碱性磷酸酶，骨桥蛋白，骨涎蛋白，骨钙素）大分子，使成纤维细胞集落区分成骨细胞集落和后者可以细分为不太成熟或更成熟的成骨细胞集落。虽然先前定义的时间分化序列的某些方面得到了证实，但从这些单细胞-单集落研究中可以明显看出几个额外的特征。首先，OB相关标志物的不同库在不同的细胞中表达，表明OB分化程序的启动变化和OB表型的异质性。其次，在基于形态学分类为成纤维细胞的集落中，异质性也很明显，并且有一些细胞表达与其为骨祖细胞一致的特征。我们的数据支持这一假设，即个别成纤维细胞和成骨细胞的标志物分子的表达是异质的。我们还得出结论，单个细胞和集落分析的聚（A）-PCR将是有用的，在替代大众扩大调查的成骨细胞分化的进展。(C)1994年出版社出版。
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 in the progression of OB differentiation. (C) 1994 Academic Press, Inc.