Turksen K, Aubin J E
Medical Research Council Group in Periodontal Physiology, University of Toronto, Ontario, Canada.
J Cell Biol. 1991 Jul;114(2):373-84. doi: 10.1083/jcb.114.2.373.
The number of identifiable stages and expression of differentiation markers in cells of the osteoblast lineage are not well understood. In the present study, a mAb, designated rat bone marrow (RBM) 211.13, was prepared that stained selectively the osteogenic and preosteoblastic cells along the surfaces of bone in calvariae, femurs, and metatarsals. The staining was cell surface associated and coincided with that for alkaline phosphatase (APase) detected histochemically. Only cells positive for APase activity by biochemical assay and not those without APase activity (e.g., fetal rat skin) stained with RBM 211.13. By immunoblotting, RBM 211.13 recognized a band coinciding with APase activity on nonreducing/nondenaturing gels, and RBM 211.13 precipitated a protein which on reduced gels migrated with an apparent molecular mass of approximately 80 kD. RBM 211.13 labeling was abolished by phosphatidylinosital-specific phospholipase C, known to release APase from the cell surface. All of these data support the concept that RBM 211.13 recognizes the bone isoenzyme of APase. RBM 211.13 was used to sort by flow cytometry the APase-positive and APase-negative cells from mixed fetal rat calvaria (RC) cell populations. The osteoprogenitors we identified earlier that form bone nodules in vitro (Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143-154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132-138) were found within the APase-positive pool. By immunopanning, RC cells were separated into APase-enriched (APase-positive, adherent) and APase-depleted (APase-negative, nonadherent) populations. The APase-positive fraction was enriched two-to-threefold for bone-forming osteoprogenitors compared to unfractionated cells, while the APase-negative population formed very few nodules under the same conditions. Both populations responded to the glucocorticoid dexamethasone (DEX) with an increase in bone nodule formation. However, the fold stimulation in bone formation in the APase-negative population was approximately 30-fold, while the fold stimulation in the APase-positive population was only approximately 5-fold. These data suggest that APase expression can be used for immunoselection to fractionate osteoblastic populations into an APase-positive population and a population initially APase-negative, that virtually all osteoprogenitors forming bone in vitro in the absence of added glucocorticoids reside in the APase-positive pool, and that the only osteoprogenitors present in the APase-negative pool are those requiring DEX to differentiate.
成骨细胞谱系细胞中可识别阶段的数量以及分化标志物的表达尚未完全明确。在本研究中，制备了一种名为大鼠骨髓（RBM）211.13的单克隆抗体，它能选择性地对颅骨、股骨和跖骨骨表面的成骨细胞和前成骨细胞进行染色。这种染色与细胞表面相关，并且与通过组织化学检测到的碱性磷酸酶（APase）的染色情况一致。只有通过生化检测显示APase活性呈阳性的细胞，而非那些无APase活性的细胞（如胎鼠皮肤），才能被RBM 211.13染色。通过免疫印迹法，RBM 211.13在非还原/非变性凝胶上识别出一条与APase活性相对应的条带，并且RBM 211.13沉淀出一种蛋白质，该蛋白质在还原凝胶上迁移时的表观分子量约为80 kD。已知磷脂酰肌醇特异性磷脂酶C可从细胞表面释放APase，用其处理后，RBM 211.13的标记被消除。所有这些数据都支持RBM 211.13识别APase骨同工酶这一概念。利用RBM 211.13通过流式细胞术从混合的胎鼠颅骨（RC）细胞群体中筛选出APase阳性和APase阴性细胞。我们之前鉴定出的能在体外形成骨结节的骨祖细胞（Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143 - 154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132 - 138）存在于APase阳性细胞群中。通过免疫淘选，将RC细胞分离为富含APase的群体（APase阳性，贴壁）和APase缺失的群体（APase阴性，不贴壁）。与未分离的细胞相比，APase阳性部分中形成骨的骨祖细胞富集了两到三倍，而在相同条件下，APase阴性群体形成的结节很少。这两个群体对糖皮质激素地塞米松（DEX）的反应都是骨结节形成增加。然而，APase阴性群体中骨形成的刺激倍数约为30倍，而APase阳性群体中骨形成的刺激倍数仅约为5倍。这些数据表明，APase表达可用于免疫分选，将成骨细胞群体分为APase阳性群体和最初APase阴性的群体，在不添加糖皮质激素的情况下，几乎所有能在体外形成骨的骨祖细胞都存在于APase阳性细胞群中，并且APase阴性细胞群中仅有的骨祖细胞是那些需要DEX才能分化的细胞。
