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细胞被分离出来的。单层培养时，细胞在生长期和融合后均呈纺锤形。Von Kossa染色阳性的细胞外基质在细胞培养中出现，融合后细胞外基质表达增加。在超微结构上，基质是电子致密的，显然是由细胞产生的。我们用流式细胞术分析了克隆性KUSA/A1细胞的细胞表面标志，以更好地表征这些细胞。C-kit、Flk-1、CD14、CD31、CD34、CD41、CD45、CD49b、CD49d、CD54、CD90、CD102、CD105、CD106、CD144、Ly-6G阴性。比较MC3T3-E1细胞的生长曲线、碱性磷酸酶(ALP)活性、体外钙化、骨钙素(骨钙素)释放和甲状旁腺素(PTH)的反应。用DNA含量测定KUSA/A1细胞的增殖率与MC3T3-E1细胞相当。处于生长期的KUSA/A1细胞的ALP活性约为MC3T3-E1细胞的10倍。体外钙化在融合前和融合后都稳步增加，融合后KUSA/A1细胞的钙化程度大约是MC3T3-E1细胞的100倍。KUSA/A1细胞的骨钙素释放在第5天达到高峰，是MC3T3-E1细胞的2~3倍。在培养过程中，KUSA/A1细胞的甲状旁腺激素反应逐渐降低，而MC3T3-E1细胞的甲状旁腺激素反应增加。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.