Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level.
Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level.
复制标题
同时检测成骨细胞分化过程中多种骨相关 mRNA 和蛋白质表达：单细胞水平的聚合酶链反应和免疫细胞化学研究。
作者：
F. Liu;L. Malaval;A. Gupta;J. Aubin
通过原位杂交和Northern分析的信使RNA表达，以及生化或免疫细胞化学分析的蛋白质表达，已经被用来研究各种成骨细胞相关分子的发育表达。这些方法表明，在体内和体外OB细胞分化的一段时间内，与OB细胞相关的大分子的表达会发生变化。然而，来自不同方法的数据和代表处于不同发育阶段的细胞的群体中的数据存在歧义。为了更准确地区分分化阶段并解决细胞间的异质性，在骨结节形成和矿化的条件下，低密度培养胎鼠颅骨细胞，并将集落形态分类为成纤维细胞或成骨细胞(早期、中期或成熟)。通过随机扩增多聚酶链式反应(PCR)对整个离散克隆和单个克隆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的表达；我们分析已知的骨相关大分子(I型胶原、碱性磷酸酶、骨桥蛋白、骨涎蛋白和骨钙素)的表达。聚合酶链式反应和免疫细胞化学显示，不同的集落类型在表达普通大分子(I型胶原)或骨相关大分子(碱性磷酸酶、骨桥蛋白、骨涎蛋白和骨钙素)方面具有可重复性，因此成纤维细胞克隆与成骨细胞克隆不同，而成骨细胞克隆又可细分为不成熟或较成熟的成骨细胞克隆。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-单集落研究中还明显地发现了几个额外的特征。首先，不同的OB相关标记在不同的细胞中表达，这表明OB分化程序的启动不同，OB表型的异质性。其次，在根据形态分类为成纤维细胞的集落中，也存在明显的异质性，并且有一些细胞表达与其为骨祖细胞的特征一致。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记分子的表达上是不同的。我们还得出结论，用Poly(A)-PCR分析单个细胞和集落将有助于替代大量群体来扩大对OB分化进展阶段的研究。
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.