Establishment of Assay System for Studying Osteoblastic Function and Differentiation Using Newly Isolated Osteoblast-Like Cell Lines

为了建立适合研究成骨细胞表型和分化的实验系统，我们从新生大鼠颅骨和人骨肉瘤中分离了多种克隆成骨样细胞系。成骨样细胞系的建立1)从大鼠颅骨中分离的大鼠成骨样细胞系罗伯- c8a、Cll、C20五种克隆成骨样细胞系。C23。C26)从1日龄大鼠颅骨中分离得到。Cll、C20和C23细胞系表达了高度的成骨特性，而C26细胞表达的成骨特性水平很低。成骨性质在C8a中的表达程度为中等。更重要的是，C26细胞不仅表现出向成骨细胞分化的潜能，还表现出向肌管细胞和脂肪细胞分化的潜能。这表明C26细胞是研究成骨细胞、肌肉细胞和脂肪细胞从未分化间充质细胞分化途径的有用细胞系。从人骨肉瘤中分离到2株克隆性成骨细胞样细胞株（YR-2和YR-6）。将这些细胞系的成骨表达与其他已建立的骨肉瘤细胞系（HOS、SAOS-2和Mg63）进行比较。本研究证明了不同的人骨肉瘤细胞系间成骨细胞表型的多样性，并提示在使用人骨肉瘤细胞系研究成骨细胞表型时，了解每个细胞系的成骨细胞特征是很重要的。骨形态发生蛋白（BMP）和视黄酸在成骨细胞分化中起重要作用。1) BMP：重组人骨形态发生蛋白-2 （rhBMP-2，由Dr. Wozney, Genetics Institute， MA， USA提供）在未成熟成骨样细胞系C26中强烈刺激成骨细胞分化并抑制成肌分化。rhBMP-2还能诱导非成骨成纤维细胞c3h101 /2细胞向成骨细胞分化。这些结果强烈提示BMP-2在未分化的间充质细胞向成骨细胞分化过程中起重要作用。2)维甲酸：维甲酸刺激C26细胞ALP活性和PTH反应性，抑制C26细胞成肌分化和脂肪分化。这些结果表明维甲酸调节未分化间充质细胞向成骨细胞、肌肉细胞和脂肪细胞的分化途径。少

In order to establish the suitable assay system for studying osteoblastic phenotypes and differentiation, we isolated various kinds of clonal osteoblast-like cell lines from neonatal rat calvariae and human osteosarcoma.I. Establishment of osteoblast-like cell lines1) Rat osteoblast-like cell lines isolated from rat calvariaeFive clonal osteoblast-like cell lines (ROB-C8a, Cll, C20. C23. C26) were isolated from 1-day old rat calvariae. Cll, C20 and C23 cell lines expressed high degree of osteoblastic properties but C26 cells expressed very low level of that properties. Expression degree of the osteoblastic properties in C8a was intermediate. More importantly, C26 cells expressed differentiation potential into not only osteoblasts but also myotubes and adipocytes. These indicated that C26 cells are useful cell line for studying the differentiation pathway of osteoblasts, muscle cells and adipocytes from undifferentiated mesenchymal cells.2) Human osteoblast-like cell lines isolated from … More human osteosarcomaTwo clonal osteoblast-like cell lines (YR-2 and YR-6) were isolated from human osteosarcoma. Osteoblastic expression of these cell lines were compared with those of other established osteosarcoma cell lines (HOS, SAOS-2 and Mg63). This study demonstrated the diversity of osteoblastic phenotypes among various human osteosarcoma cell lines tested, and suggested that much understanding of the osteoblastic characteristics in each cell line is important when human osteosarcoma cell lines are used for studying osteoblastic phenotypes.II. Regulatory mechanism of osteoblast differentiationWe demonstrated that bone morphogenetic protein (BMP) and retinoic acid play an important role in osteoblast differentiation.1) BMP : Recombinant human bone morphogenetic protein-2 (rhBMP-2 ; provided from Dr. Wozney, Genetics Institute, MA, USA) strongly stimulated osteoblastic differentiation and inhibited myogenic differentiation in the immature osteoblast-like cell line, C26. rhBMP-2 also induced non-osteogenic fibroblasts, C3H1OT1/2 cells, to differentiate into osteoblastic cells. These results strongly suggest that BMP-2 plays an important role in osteoblastic differentiation from undifferentiated mesenchymal cells.2) Retinoic acid : Retinoic acid stimulated ALP activity and PTH responsiveness, and inhibited myogenic and adipose differentiation in C26 cells. These results suggest that retinoic acid regulates differentiation pathway into osteoblasts, muscle cells and adipocytes from undifferentiated mesenchymal cells. Less