Satomura K, Derubeis A R, Fedarko N S, Ibaraki-O'Connor K, Kuznetsov S A, Rowe D W, Young M F, Gehron Robey P
Craniofacial and Skeletal Diseases Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Cell Physiol. 1998 Dec;177(3):426-38. doi: 10.1002/(SICI)1097-4652(199812)177:3<426::AID-JCP6>3.0.CO;2-F.
Bone marrow stromal cells (BMSCs) are a heterogeneous population of cells derived from colony-forming units-fibroblastic (CFU-Fs). These cells reside in the bone marrow cavity and are capable of differentiating into several cell phenotypes including osteoblasts, chondroblasts, hematopoiesis-supporting stromal cells, and adipocytes. However, the factors that regulate the proliferation and differentiation of the BMSC population are for the most part unknown. Since many members of the receptor tyrosine kinase (RTK) family have been shown to participate in growth control of various mesenchymal cell populations, in this study we examined the expression and function of RTKs in the BMSC population. Degenerate oligonucleotides corresponding to two conserved catalytic domains of the RTK family and RT-PCR were used initially to determine which RTKs are expressed in the human BMSC (hBMSC) system. After subcloning the amplification product generated from mRNA of a multicolony-derived hBMSC strain, PDGF receptor (beta), EGF receptor, FGF receptor 1, and Axl were identified by DNA sequencing of 26 bacterial colonies. Furthermore, PDGF and EGF were found to enhance BMSC growth in a dose-dependent manner and to induce tyrosine phosphorylation of intracellular molecules, including the PDGF and EGF receptors themselves, demonstrating the functionality of these receptors. On the other hand, bFGF was found to have little effect on proliferation or tyrosine phosphorylation. Since single colony-derived hBMSC strains are known to vary from one colony to another in colony habit (growth rate and colony structure) and the ability to form bone in vivo, the expression levels of these RTKs were determined in 18 hBMSC clonal strains by semiquantitative RT-PCR and were found to vary from one clonal strain to another. While not absolutely predictive of the osteogenic capacity of individual clonal strains, on average, relatively high levels of PDGF-receptor were found in bone-forming strains, while on average, nonbone-forming strains had relatively high levels of EGF-receptor. Taken together, these results indicate that RTKs play a role in the control of hBMSC proliferation, and that the differential pattern of RTK expression may be useful in correlating the biochemical properties of individual clonal strains with their ability to produce bone in vivo.
骨髓基质细胞（BMSCs）是源自成纤维细胞集落形成单位（CFU-Fs）的异质性细胞群体。这些细胞存在于骨髓腔中，能够分化为多种细胞表型，包括成骨细胞、软骨细胞、支持造血的基质细胞和脂肪细胞。然而，调节BMSC群体增殖和分化的因素在很大程度上尚不清楚。由于受体酪氨酸激酶（RTK）家族的许多成员已被证明参与各种间充质细胞群体的生长控制，在本研究中，我们检测了RTKs在BMSC群体中的表达和功能。最初使用与RTK家族两个保守催化结构域相对应的简并寡核苷酸和RT-PCR来确定哪些RTKs在人BMSC（hBMSC）系统中表达。在对多克隆来源的hBMSC菌株的mRNA产生的扩增产物进行亚克隆后，通过对26个细菌菌落进行DNA测序鉴定出了血小板衍生生长因子受体（β）、表皮生长因子受体、成纤维细胞生长因子受体1和Axl。此外，发现血小板衍生生长因子（PDGF）和表皮生长因子（EGF）以剂量依赖的方式促进BMSC生长，并诱导细胞内分子的酪氨酸磷酸化，包括PDGF和EGF受体本身，证明了这些受体的功能。另一方面，发现碱性成纤维细胞生长因子（bFGF）对增殖或酪氨酸磷酸化几乎没有影响。由于已知单克隆来源的hBMSC菌株在菌落习性（生长速率和菌落结构）以及体内成骨能力方面因菌落不同而有所差异，通过半定量RT-PCR测定了18个hBMSC克隆菌株中这些RTKs的表达水平，发现其在不同克隆菌株之间有所不同。虽然不能绝对预测单个克隆菌株的成骨能力，但平均而言，在成骨菌株中发现血小板衍生生长因子受体水平相对较高，而在非成骨菌株中，表皮生长因子受体水平相对较高。综上所述，这些结果表明RTKs在hBMSC增殖控制中发挥作用，并且RTK表达的差异模式可能有助于将单个克隆菌株的生化特性与其体内成骨能力相关联。
