Identification of osteogenic factor (5) in the KUSA-A1 osteoblasts

KUSA/A1细胞最初是作为体内诱导造血的细胞分离的。单层培养时，细胞在生长期和汇合后呈梭形。细胞培养中出现vonKossa染色阳性的细胞外基质，汇合后增加。在超微结构上，基质是电子致密的，并且清楚地由细胞产生。我们通过流式细胞术分析了克隆KUSA/A1细胞上的细胞表面标志物，以更好地表征这些细胞。发现细胞对Sca-1、CD 44、Ly-6C和CD 140呈强阳性（比同种型对照高10倍以上），对CD 29呈弱阳性，对c-kit、Flk-1、CD 14、CD 31、CD 34、CD 41、CD 45、CD 49 b、CD 49 d、CD 54、CD 90、CD 102、CD 105、CD 106、CD 144和Ly-6 G呈阴性。以MC 3 T3-E1细胞为对照，分析其生长曲线、ALP活性、体外钙化、骨钙素（骨钙素）释放和对PTH的反应。KUSA/A1细胞的增殖能力与MC 3 T3-E1细胞相当。KUSA/A1细胞在生长期的ALP活性比MC 3 T3-E1细胞高约10倍。在体外钙化，这稳步增加之前和之后的融合，是约100倍以上的融合阶段后，在KUSA/A1细胞比MC 3 T3-E1细胞。在KUSA/A1细胞中，骨钙素释放到培养基中在第5天达到峰值，并且比MC 3 T3-E1细胞高2至3倍。在培养过程中，KUSA/A1细胞的PTH反应（以cAMP的产生来衡量）逐渐下降，而MC 3 T3-E1细胞的PTH反应则逐渐增加。这种对PTH的反应不需要任何诱导，并且与培养条件无关。

KUSA/A1 cells were originally isolated as cells which induce hematopoiesis in vivo. When cultured in monolayer, the cells were spindle-shaped in the growth phase and after confluence. Extracellular matrix positive for von Kossa stain appeared in cell culture and increased after confluence. Ultrastructurally, the matrix was electron dense and was clearly produced by the cells. We analyzed the cell surface markers on the clonal KUSA/A1 cells by flow cytometry to better characterize these cells. The cells were found to be strongly positive (more than 10-fold greater than the isotype control) for Sca-1, CD44, Ly-6C and CD140, weakly positive for CD29, and negative for c-kit, Flk-1, CD14, CD31, CD34, CD41, CD45, CD49b, CD49d, CD54, CD90, CD102, CD105, CD106, CD144 and Ly-6G. We then analyzed growth curve, ALP activity, in vitro calcification, osteocalcin (bone gla protein) release, and response to PTH, in comparison with MC3T3-E1 cells. KUSA/A1 proliferation, which was measured by DNA content, was equivalent to that of MC3T3-E1 cells. ALP activity of KUSA/A1 cells in growth phase was approximately 10-fold higher than in MC3T3-E1 cells. In vitro calcification, which steadily increased both before and after confluence, was approximately 100-fold higher after the confluent stage in KUSA/A1 cells than in MC3T3-E1 cells. Osteocalcin release into culture media peaked on day 5 in KUSA/A1 cells and was 2 to 3-fold higher than in MC3T3-E1 cells. PTH response, measured as cAMP production, gradually decreased during the culture period in KUSA/A1 cells, but increased in MC3T3-E1 cells. This response to PTH did not require any induction and was independent of culture conditions.