McCarthy T L, Centrella M, Canalis E
Department of Medicine, (Endocrine Section), and Research Laboratory, Saint Francis Hospital and Medical Center, Hartford, CT 06105.
J Bone Miner Res. 1988 Aug;3(4):401-8. doi: 10.1002/jbmr.5650030406.
Primary bone cell cultures are used widely to examine the regulation of bone metabolism by growth factors and hormones. Characterization of this model system is now being conducted at the molecular level to define modulation of gene expression. Cells were obtained from rat parietal bone by sequential collagenase digestions. Cell populations were evaluated for bone-related products, including collagen isoform expression and mRNA levels, alkaline phosphatase activity, and osteocalcin production. Serum-deprived, confluent cultures of the first and second collagenase-released populations produced a lower percentage of total protein as collagen than the third, fourth, and fifth populations, while co-culturing the third through fifth populations resulted in the highest level. Collagen typing on SDS-polyacrylamide gels revealed an abundance of mature type I collagen in all cell populations; type III collagen synthesis was undetectable by this method. This is in contrast to the presence of cytoplasmic mRNA for both type I and type III collagen in all cell populations, suggesting post-transcriptional modulation of type III collagen synthesis. The expression of alkaline phosphatase and osteocalcin was highest in cultures of later released cells, indicating that these cell populations display phenotypic characteristics associated with cells of the osteoblast lineage.
原代骨细胞培养被广泛用于研究生长因子和激素对骨代谢的调节。目前正在分子水平对该模型系统进行表征，以确定基因表达的调控。通过连续胶原酶消化从大鼠顶骨获取细胞。对细胞群体进行与骨相关产物的评估，包括胶原异构体表达和mRNA水平、碱性磷酸酶活性以及骨钙素生成。与第三、第四和第五群体相比，血清饥饿的第一和第二胶原酶释放群体的汇合培养物中，作为胶原的总蛋白百分比更低，而第三至第五群体的共培养产生的水平最高。SDS-聚丙烯酰胺凝胶上的胶原分型显示，所有细胞群体中都有大量成熟的I型胶原；用该方法未检测到III型胶原的合成。这与所有细胞群体中I型和III型胶原的细胞质mRNA的存在形成对比，表明III型胶原合成存在转录后调控。碱性磷酸酶和骨钙素的表达在后期释放细胞的培养物中最高，表明这些细胞群体表现出与成骨细胞谱系细胞相关的表型特征。
